Gene therapy for aadc deficiency

ABSTRACT

The present invention is directed to compositions and methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency. This invention includes a method of treating AADC deficiency in a pediatric subject, comprising the steps of: (a) providing a pharmaceutical formulation comprising an rAAV2-hAADC vector, (b) stereotactically delivering the pharmaceutical formulation to at least one target site in the brain of the subject in a dose of an amount at least about 1.8×1011 vg; wherein delivering the pharmaceutical formulation to the brain is optionally by frameless stereotaxy, and optionally wherein the dose is an amount of at least about 2.4×1011 vg and in some embodiments wherein the pharmaceutical formulation comprises a rAAV2-hAADC vector concentration of about 5.7×1011 vg/mL. This invention is also directed to methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency, wherein the method optionally further comprises the step of administering a therapeutically effective dose of dopamine-antagonist to the subject such as risperidone. This invention is also directed to methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency, wherein the method optionally comprises providing a pharmaceutical formulation comprising an rAAV2-hAADC vector, and empty capsids.

FIELD OF THE INVENTION

This invention concerns a method of treating AADC deficiency in asubject, comprising the steps of (a) providing a pharmaceuticalformulation comprising an rAAV2-hAADC vector and (b) delivering thepharmaceutical formulation to the brain of the subject; in an amount ofat least about 1.8×10¹¹ vg. Particular reference is made to treatingpediatric subjects using frameless stereotaxy, treating dyskinesia inpediatric subjects. Particular reference is also made to pharmaceuticalformulations comprising rAAV2-hAADC vectors and empty capsids.

REFERENCE TO SEQUENCE LISTING

A paper copy of the sequence listing and a computer readable form of thesame sequence listing are appended below and herein incorporated byreference. The information recorded in computer readable form isidentical to the written sequence listing, according to 37 C.F.R.1.821(f).

BACKGROUND

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare geneticdisorder believed to arise from mutation of the DDC gene (dopadecarboxylase). Without being bound by any theory, AADC is an enzymereported in the literature as responsible for the final step in thesynthesis of neurotransmitters dopamine (which is then synthesized intonorepinephrine and subsequently epinephrine) and serotonin (which isthen synthesized into melatonin). AADC deficiency results in severedevelopmental failures, global muscular hypotonia and dystonia, severe,long-lasting seizures known as oculo-gyric crises, frequenthospitalizations (including prolonged stays in intensive care), and theneed for life-long care. Symptoms and severity vary depending on thetype of underlying genetic mutation which abrogates AADC enzymefunction.

Presently, treatment options are limited. Only patients with relativelymild forms of the disease respond to drugs. Furthermore, patients obtainrelief from only a limited subset of symptoms. Drug therapy provideslittle or no benefit for many patients who often die during childhood.Patients with severe forms usually die before the age of 6 or 7 yearsdue to severe motor dysfunction, autonomic abnormalities, and secondarycomplications such as choking, hypoxia, and pneumonia.

Only limited research has been directed toward AADC deficiency inchildren. Only one group has reported the restoration of some motordevelopment and function in four children treated for AADC deficiency bygene therapy using a low dosage. No dose has been used above 1.6×10¹¹ vgper subject. See Wuh-Liang Hwu, et al., “Gene Therapy for Aromatic1-Amino Acid Decarboxylase Deficiency,” Sci. Transl. Med. 4, 134 (2012),and Wuh Liang Hwu, et al., U.S. Pat. App. Pub. No. US 2012/0220648.These references and all publications cited herein are incorporated byreference in their entirety.

Other research has been limited to adult patients with Parkinson'sdisease. Clinical studies using gene therapy in Parkinson's disease haveshown that the adeno-associated virus (AAV) type 2 vector-mediateddelivery of the human AADC gene (hAADC) into the putamen is safe andwell tolerated in adults. See C. W. Christine, et al., “Safety andtolerability of putaminal AADC gene therapy for Parkinson disease,”Neurology 73, 1662-1669 (2009); and S. Muramatsu, et al., “A phase Istudy of aromatic L-amino acid decarboxylase gene therapy forParkinson's disease,” Mol. Ther. 18, 1731-1735 (2010); K. Ozawa et al.U.S. Pat. No. 7,588,757 “Methods of treating Parkinson's disease usingrecombinant adeno-associated virus virions”; and K. Bankiewicz et al.,U.S. Pat. No. 6,309,634, “Methods of treating Parkinson's disease usingrecombinant adeno-associated vector (rAAV).”

Several problems of AADC gene therapy treatments need to be addressed.(i) There is no data as to the safety or efficacy of high doses inchildren. (ii) There is no data as to the safety or efficacy of dosesselected according to a child's age. (iii) Stereotaxy for AADC genetherapy in Parkinson's disease uses a cumbersome skull-fixed head framethat is not feasible for pediatric subjects. (iv) There is no data as tothe safety or efficacy of treatments for pediatric subjects experiencingAADC gene therapy induced dyskinesia. (v) A subject's immune systemlimits AADC gene transduction. Without being bound by theory, it isbelieved that macrophages neutralize adeno-associated virus(AAV)-mediated gene delivery by phagocytosis of AAV particles.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods fortreating aromatic L-amino acid decarboxylase (AADC) deficiency.

For Summery of Invention

This invention includes a method of treating AADC deficiency in apediatric subject, comprising the steps of: (a) providing apharmaceutical formulation comprising an rAAV2-hAADC vector, (b)stereotactically delivering the pharmaceutical formulation to at leastone target site in the brain of the subject in a dose of an amount atleast about 1.8×10¹¹ vg; wherein delivering the pharmaceuticalformulation to the brain is by frameless stereotaxy, and optionallywherein the dose is an amount of at least about 2.4×10¹¹ vg and in someembodiments wherein the pharmaceutical formulation comprises arAAV2-hAADC vector concentration of about 5.7×10¹¹ vg/mL. Particularnote is made that the pharmaceutical formulation is delivered at a rateof about 3 μL/min and further wherein the pharmaceutical formulation isdelivered to at least one target site in a brain at a dose volume ofabout 80 μL per target site.

Particular note is also made that the pharmaceutical formulation isdelivered to a putamen of the brain. In one embodiment, thepharmaceutical formulation is delivered bilaterally to each putamen. Inone particular embodiment, said bilateral delivery is to points about 1mm to about 10 mm apart. Optionally the rAAV2-hAADC vector comprises:(a) a WT AAV2 capsid, and (b) a recombinant DNA DDC gene insertcomprising: (i) a first inverted terminal repeat (ITR), (ii) acytomegalovirus (CMV) immediate early promoter (IEP)IEP, (iii) a humanβ-globin partial intron2/exon 3, (iv) a nucleic acid sequence encodinghAADC, (v) an SV40 poly A tail, and (vi) a second ITR; wherein the firstITR and second ITR flank the CMV IEP promoter and the Poly A tail.Particular note is made that the nucleic acid sequence encoding hAADC isan unmodified DDC cDNA. In further embodiments, the method furthercomprises the step of: (c) administering a therapeutically effectivedose of dopamine-antagonist to the subject. Particular note is made thatthe dopamine-antagonist can be optionally clozapine, haloperidol,olanzapine paliperidone, quetiapine, risperidone, or ziprasidone,optionally administered at a dose from about 0.1 mg daily to about 1000mg daily.

This invention also includes a method of treating g AADC deficiency in apediatric subject, comprising the steps of: (a) providing apharmaceutical formulation comprising an rAAV2-hAADC vector, and (b)stereotactically delivering the pharmaceutical formulation to at leastone target site in the brain of the subject in a dose of an amount atleast about 2.4×10¹¹ vg. In some embodiments wherein the pharmaceuticalformulation comprises a rAAV2-hAADC vector concentration of about7.5×10¹¹ vg/mL. Particular note is made that the pharmaceuticalformulation is delivered at a rate of about 3 μL/min and further whereinthe pharmaceutical formulation is delivered to at least one target sitein a brain at a dose volume of about 80 μL per target site. Particularnote is also made that the pharmaceutical formulation is delivered to aputamen of the brain. In one embodiment, the pharmaceutical formulationis delivered bilaterally to each putamen. In one particular embodiment,said bilateral delivery is to points about 1 mm to about 10 mm apart.Optionally the rAAV2-hAADC vector comprises: (a) a WT AAV2 capsid, and(b) a recombinant DNA DDC gene insert comprising: (i) a first invertedterminal repeat (ITR), (ii) a cytomegalovirus (CMV) immediate earlypromoter (IEP)IEP, (iii) a human β-globin partial intron2/exon 3, (iv) anucleic acid sequence encoding hAADC, (v) an SV40 poly A tail, and (vi)a second ITR; wherein the first ITR and second ITR flank the CMV IEPpromoter and the Poly A tail. Particular note is made that the nucleicacid sequence encoding hAADC is an unmodified DDC cDNA. In furtherembodiments, the method further comprises the step of: (c) administeringa therapeutically effective dose of dopamine-antagonist to the subject.Particular note is made that the dopamine-antagonist can be optionallyclozapine, haloperidol, olanzapine, paliperidone, quetiapine,risperidone, or ziprasidone, optionally administered at a dose fromabout 0.1 mg daily to about 1000 mg daily.

This invention also includes a method of treating AADC deficiency in apediatric subject aged less than about 3 years, comprising the steps of:(a) providing a pharmaceutical formulation comprising an rAAV2-hAADCvector, and (b) stereotactically delivering the pharmaceuticalformulation to at least one target site in the brain of the subject in adose of an amount at least about 2.0×10¹¹ vg; and optionally wherein thedose is an amount of at least about 2.4×10¹¹ vg and in some embodimentswherein the pharmaceutical formulation comprises a rAAV2-hAADC vectorconcentration of about 7.5×10¹¹ vg/mL. Particular note is made that thepharmaceutical formulation is delivered at a rate of about 3 μL/min andfurther wherein the pharmaceutical formulation is delivered to at leastone target site in a brain at a dose volume of about 80 μL per targetsite. Particular note is also made that the pharmaceutical formulationis delivered to a putamen of the brain. In one embodiment, thepharmaceutical formulation is delivered bilaterally to each putamen. Inone particular embodiment, said bilateral delivery is to points about 1mm to about 10 mm apart. Optionally the rAAV2-hAADC vector comprises:(a) a WT AAV2 capsid, and (b) a recombinant DNA DDC gene insertcomprising: (i) a first inverted terminal repeat (ITR), (ii) acytomegalovirus (CMV) immediate early promoter (IEP)IEP, (iii) a humanβ-globin partial intron2/exon 3, (iv) a nucleic acid sequence encodinghAADC, (v) an SV40 poly A tail, and (vi) a second ITR; wherein the firstITR and second ITR flank the CMV IEP promoter and the Poly A tail.Particular note is made that the nucleic acid sequence encoding hAADC isan unmodified DDC cDNA. In further embodiments, the method furthercomprises the step of: (c) administering a therapeutically effectivedose of dopamine-antagonist to the subject. Particular note is made thatthe dopamine-antagonist can be optionally clozapine, haloperidol,olanzapine, paliperidone, quetiapine, risperidone, or ziprasidone,optionally administered at a dose from about 0.1 mg daily to about 1000mg daily.

This invention also includes a method of treating AADC deficiency in apediatric subject aged about 3 or more years, comprising the steps of:(a) providing a pharmaceutical formulation comprising an rAAV2-hAADCvector, and (b) stereotactically delivering the pharmaceuticalformulation to at least one target site in the brain of the subject in adose from about 1.8×10¹¹ vg to about 2.4×10¹¹ vg. Particular note ismade that the pharmaceutical formulation is delivered at a rate of about3 μL/min and further wherein the pharmaceutical formulation is deliveredto at least one target site in a brain at a dose volume of about 80 μLper target site. Particular note is also made that the pharmaceuticalformulation is delivered to a putamen of the brain. In one embodiment,the pharmaceutical formulation is delivered bilaterally to each putamen.In one particular embodiment, said bilateral delivery is to points about1 mm to about 10 mm apart. Optionally the rAAV2-hAADC vector comprises:(a) a WT AAV2 capsid, and (b) a recombinant DNA DDC gene insertcomprising: (i) a first inverted terminal repeat (ITR), (ii) acytomegalovirus (CMV) immediate early promoter (IEP)IEP, (iii) a humanβ-globin partial intron2/exon 3, (iv) a nucleic acid sequence encodinghAADC, (v) an SV40 poly A tail, and (vi) a second ITR; wherein the firstITR and second ITR flank the CMV IEP promoter and the Poly A tail.Particular note is made that the nucleic acid sequence encoding hAADC isan unmodified DDC cDNA. In further embodiments, the method furthercomprises the step of: (c) administering a therapeutically effectivedose of dopamine-antagonist to the subject. Particular note is made thatthe dopamine-antagonist can be optionally clozapine, haloperidol,olanzapine paliperidone, quetiapine risperidone, or ziprasidone,optionally administered at a dose from about 0.1 mg daily to about 1000mg daily.

This invention also includes a method of treating AADC deficiency in apediatric subject, comprising the steps of: (a) providing apharmaceutical formulation comprising an AAV2-hAADC vector, (b)delivering the pharmaceutical formulation to the brain of the subject,and (c) administering a therapeutically effective dopamine-antagonist tothe subject. The dopamine-antagonist is optionally administered fromabout the beginning of week-4 after gene-transduction until at leastabout the end of 12-weeks after gene-transduction. Particular note ismade that the dopamine-antagonist can be optionally clozapine,olanzapine paliperidone, quetiapine, risperidone, or ziprasidone.Particular note is made that the dopamine-antagonist is optionallyadministered at a dose from about 0.1 mg daily to about 1000 mg daily.

This invention also includes a pharmaceutical formulation comprising:(a) an rAAV2 hAADC vector, and (b) 1×PBS. The pharmaceutical formulationoptionally further comprises: (c) about 200 mM NaCl. The pharmaceuticalformulation optionally further comprises rAAV2 hAADC vector at aconcentration of about 5.7×10¹¹ vg/mL. Particular note is made that thepharmaceutical formulation optionally further comprises: (d) emptycapsids. Particular note is made that the pharmaceutical formulationoptionally comprises empty capsids at a percentage of at least about0.1% cp/cp.

In one aspect, the invention is directed to a method of treatingpediatric AADC deficiency in a subject, comprising the steps of: (a)providing a pharmaceutical formulation comprising an rAAV2-hAADC vector,and (b) delivering the pharmaceutical formulation to the brain of thesubject; wherein: (i) the pharmaceutical formulation is delivered at adose of at least about 1.8×10¹¹ vg, and (ii) the pharmaceuticalformulation is delivered using a frameless stereotactic procedure.

Optionally, the dose is at least about 2.4×10¹¹ vg, at least about1×10¹² vg, at least about 1×10¹⁴ vg, or at least about 1×10¹⁶ vg.Preferably the dose ranges from about 1.8×10¹¹ vg to about 1.5×10¹² vg,or ranges from about 1.8×10¹¹ vg to about 2.4×10¹¹ vg. More preferably,the dose is about 1.8×10¹¹ vg, about 2.4×10¹¹ vg, or about 1.5×10¹² vg.

Optionally, the pharmaceutical formulation is delivered at a rateranging from of about 0.1 μL/min to about 10 μL/min, or at a rateranging from about 1 μL/min to about 5 μL/min, or at a rate ranging fromabout 2 μL/min to about 3 μL/min. Preferably, the pharmaceuticalformulation is delivered at a rate of 1 μL/min, 2 μL/min, 3 μL/min, 4μL/min, 5 μL/min, 6 μL/min, 7 μL/min, 8 μL/min 9 μL/min, or 10 μL/min.More preferably, the pharmaceutical formulation is delivered at a rateof 3 μL/min.

Optionally, the pharmaceutical formulation is delivered at a dose volumeranging from about 1 μL to about 1000 μL per target site, at a dosevolume ranging from about 10 μL to about 100 μL per target site, or at adose volume ranging from about 50 μL to about 100 μL per target site.Preferably, the pharmaceutical formulation is delivered at a dose volumeof about 80 per target site.

In one embodiment, the pharmaceutical formulation, comprising arAAV2-hAADC vector concentration of 5.7×10¹¹ vg/mL, is delivered to at adose volume of about 80 μL per target site at a rate of about 3 μL/min.

In one embodiment, the rAAV2-hAADC vector comprises: (a) a WT AAV2capsid, and (b) a recombinant DNA DDC gene insert comprising: (i) afirst AAV2 ITR (ii) a cytomegalovirus (CMV) immediate early promoter(IEP), (iii) a human β-globin partial intron2/exon 3, (iv) a nucleicacid sequence encoding hAADC, (v) a Simian vacuolating virus 40 (SV40)poly A tail, and (vi) a second AAV2 ITR. Preferably, the first andsecond ITRs flank the other genetic elements of the gene insert.Preferably, the nucleic acid sequence encoding hAADC is an unmodifiedDDC cDNA.

In one embodiment, the pharmaceutical formulation is delivered to aputamen of the subject, or preferably both putamen of the subject.

Optionally, the pharmaceutical formulation is delivered bilaterally(i.e., to both putamens of the subject). Preferably, the pharmaceuticalformulation is delivered to four target points wherein each putamencontains two target points separated dorsolaterally. More preferably,the pharmaceutical formulation is delivered to a deep target point and ashallow target point in each putamen. Optionally, the deep target pointis from about the center to about 5 mm from the center of the putamen,or from about the center to about 5 mm from the center of the putamen.Optionally, the shallow target point is from about 10 mm to about 1 mmfrom the surface of the putamen. Optionally the deep target point isabout 5 mm from the center of the putamen. Optionally, the shallowtarget point is about 5 mm from the surface of the putamen.

Preferably, the two target points are sufficiently distant to each otherin the dorsolateral direction, as confirmed by computed tomography (CT)and magnetic resonance imaging (MRI). The stratum is shaped like anellipse, with a long axis anterior-posterior. The upper half of thestratum is dorsal lateral. The long axis is divided into three sections.The target points are set as the middle two points between the sections.Entry points on the skull and trajectories for injection are drawn sothat the catheter will not pass through a blood vessel. The catheter isinserted to a distance about 2 mm away from the target point. Theinfusion is started. During the infusion, the catheter is drawn backgradually until a distance about 2 mm beneath the margin of the putamen.In one embodiment, the catheter is drawn back at any rate ranging fromabout 0.1 mm/min to about 2 mm/min. In one embodiment, the catheter isdrawn back at a rate of about 0.2 mm/min. In another embodiment, thecatheter is drawn back at a rate of about 0.3 mm/min. In anotherembodiment, the catheter is drawn back at a rate of about 0.4 mm/min. Inanother embodiment, the catheter is drawn back at a rate of about 0.5mm/min.

Optionally, the two target points are spaced from about 1 mm to about 10mm apart, or are spaced from about 2 mm to about 5 mm apart. Optionally,the two target points are spaced about 5 mm apart.

In one embodiment, the method uses a frameless guidance system which ismodified for use in children.

In one embodiment, the AAV2-hAADC vector is delivered bilaterally toshallow and deep target points in a subject's putamen. In oneembodiment, the method is directed to treating children ranging in agefrom about 2 to about 8.

Another aspect the invention is directed to a method of treatingpediatric AADC deficiency in a subject aged less than about 3 years,comprising the steps of: (a) providing a pharmaceutical formulationcomprising an AAV2-hAADC vector; (b) delivering the pharmaceuticalformulation to the brain of the subject; wherein: (i) the pharmaceuticalformulation is delivered at a dose of at least about 2×10¹¹ vg, and (ii)the pharmaceutical formulation is delivered using a framelessstereotactic procedure. Optionally, the subject is aged less than about3 years, aged less than about 2 years, aged less than about 1 year, oraged less than about 6 months. Optionally, the dose is at least about2×10¹¹ vg, at least about 2.4×10¹¹ vg, at least about 5×10¹¹ vg, atleast about 1×10¹² vg, at least about 2×10¹¹ vg, or at least about2×10¹² vg. Preferably the dose is about 2.4×10¹¹ vg per subject.

Another aspect the invention is directed to a method of treatingpediatric AADC deficiency in a subject aged about 3 or more years,comprising the steps of: (a) providing a pharmaceutical formulationcomprising an AAV2-hAADC vector; (b) delivering the pharmaceuticalformulation to the brain of the subject; wherein: (i) the pharmaceuticalformulation is delivered at a dose from about 1.8×10¹¹ vg to about2.4×10¹¹ vg, and (ii) the pharmaceutical formulation is delivered usinga frameless stereotactic procedure. Optionally, the dose is about1.8×10¹¹ vg or about 2.4×10¹¹ vg per subject. Preferably, the dose isabout 1.8×10¹¹ vg per subject.

Optionally, the frameless trajectory-based stereotactic procedurecomprises: (a) installing one or more bone fiducial markers, (b)installing a skull-mounted platform, (c) drilling a burr hole in a sideof the skull, (d) locating a target point guided by MRI and CT, (e)inserting a guide tube and stylet to about 2 cm from the target point,(f) removing the stylet and inserting a catheter, and (g) infusing thepharmaceutical formulation. Preferably, the frameless trajectory-basedstereotactic procedure further comprises: (h) locating a second targetpoint guided by MRI and CT, (i) inserting a guide tube and stylet toabout 2 cm from the second target point, (j) removing the stylet andinserting a catheter, and (k) infusing the pharmaceutical formulation.More preferably, the step of infusing the pharmaceutical formulationcomprises withdrawing the catheter allowing the pharmaceuticalformulation to be distributed along a tract of about 4 mm to about 8 mm.

Optionally, the fiducial markers comprise stainless steel, titanium orgold. Preferably, the fiducial markers comprise stainless steel.

Optionally, at least about 2, at least about 3, at least about 4, atleast about 5, at least 6, at least about 7, at least about 8, at least9, at least about 10 fiducial markers are installed. Preferably, atleast about 4 fiducial markers are installed. More preferably, at leastabout 8 fiducial markers are installed. Optionally, 2, 3, 4, 5, 6, 7, 8,9 or 10 fiducial markers are installed. In one embodiment, 8 fiducialmarkers are installed in a circle on the lower part of the skull bones.In another embodiment, 7 fiducial markers are installed in a circle onthe lower part of the skull bones. In another embodiment, 6 fiducialmarkers are installed in a circle on the lower part of the skull bones.In another embodiment, 5 fiducial markers are installed in a circle onthe lower part of the skull bones. In another embodiment, 4 fiducialmarkers are installed in a circle on the lower part of the skull bones.Without being bound by theory, the fiducial markers are installed on thelower part of the skull bones because the lower part of the skull inthicker.

Another aspect the invention is directed to a method of treatingpediatric AADC deficiency in a subject, comprising the steps of: (a)providing a pharmaceutical formulation comprising an AAV2-hAADC vector,(b) delivering the pharmaceutical formulation to the brain of thesubject, and (c) administering a dopamine-antagonist to the subject.

Optionally, the dopamine-antagonist is administered simultaneously withgene therapy, is administering beginning at emergence of dyskinesiasymptoms, or is administering beginning at about the beginning of week-4after gene-transduction. Optionally, the dopamine-antagonist isadministered for a duration ranging from about 1 week to about 12 weeks.Optionally, the dopamine-antagonist is administered until at least theend of about 4-weeks after gene-transduction, until at least the end ofabout 6-weeks after gene-transduction, until at least the end of about8-weeks after gene-transduction, until at least the end of about10-weeks after gene-transduction, or until at least the end of about12-weeks after gene-transduction. Optionally, the dopamine-antagonist isadministered until the subject no longer exhibits symptoms ofdyskinesia. Preferably, the dopamine-antagonist is administered fromabout the beginning of week-4 until about the end of 12-weeks aftergene-transduction.

Optionally, the dopamine-antagonist is clozapine, haloperidol,olanzapine paliperidone, quetiapine risperidone, or ziprasidone.Preferably, the dopamine-antagonist is haloperidol or risperidone. Morepreferably, the dopamine-antagonist is risperidone.

Optionally, the dopamine-antagonist is administered at a dose from about0.01 mg daily to about 1000 mg daily, from about 1 mg daily to about 10mg daily, or from about 0.1 mg daily to about 1 mg daily. In oneembodiment, the dopamine-antagonist is administered at a dose of about0.1 mg per day. In another embodiment, the dopamine-antagonist isadministered at a dose of about 0.2 mg per day. In another embodiment,the dopamine-antagonist is administered at a dose of about 0.3 mg perday. In another embodiment, the dopamine-antagonist is administered at adose of about 0.4 mg per day. In another embodiment, thedopamine-antagonist is administered at a dose of about 0.5 mg per day.In another embodiment, the dopamine-antagonist is administered at a doseof about 0.6 mg per day. In another embodiment, the dopamine-antagonistis administered at a dose of about 0.7 mg per day. In anotherembodiment, the dopamine-antagonist is administered at a dose of about0.8 mg per day. In another embodiment, the dopamine-antagonist isadministered at a dose of about 0.9 mg per day. In another embodiment,the dopamine-antagonist is administered at a dose of about 1.0 mg perday.

In one embodiment, risperidone is administered at a dose of about 0.1 mgper day. In another embodiment, risperidone is administered at a dose ofabout 0.2 mg per day. In another embodiment, risperidone is administeredat a dose of about 0.3 mg per day. In another embodiment, risperidone isadministered at a dose of about 0.4 mg per day. In another embodiment,risperidone is administered at a dose of about 0.5 mg per day. Inanother embodiment, risperidone is administered at a dose of about 0.6mg per day. In another embodiment, risperidone is administered at a doseof about 0.7 mg per day. In another embodiment, risperidone isadministered at a dose of about 0.8 mg per day. In another embodiment,risperidone is administered at a dose of about 0.9 mg per day. Inanother embodiment, risperidone is administered at a dose of about 1.0mg per day.

In one particular embodiment, 0.1 mL of a 1 mg/mL oral solution ofrisperidone is administered twice daily (BID). In another particularembodiment, 0.2 mL of a 1 mg/mL oral solution of risperidone isadministered twice daily.

In one embodiment, the frameless system comprises: installing one ormore bone fiducial markers, installing a skull-mounted platform,drilling a burr hole in a side of the skull, locating a target pointguided by MRI and CT, inserting a guide tube and stylet toward thetarget point, removing the stylet and inserting a catheter, and infusingthe pharmaceutical formulation.

In another aspect, the invention is directed to a recombinant AAV2-hAADCvector comprising an AAV2 capsid, and a DDC gene insert. In oneembodiment, the gene insert comprises: two AAV2 ITRs flanking a CMV IEPpromoter, a human β-globin intron-2/exon-3 enhancer, a nucleic acidsequence encoding hAADC, and a SV40 poly A tail. In one embodiment, therAAV2-hAADC vector comprises: (a) a WT AAV2 capsid, and (b) arecombinant DNA DDC gene insert comprising: (i) a first AAV2 ITR (ii) aCMV IEP promoter, (iii) a human β-globin partial intron 2/exon 3, (iv) anucleic acid sequence encoding hAADC, (v) an SV40 poly A tail, and (vi)a second AAV2 ITR. Preferably, the first and second ITRs flank the othergenetic elements of the gene insert. Preferably, the nucleic acidsequence encoding hAADC is an unmodified DDC cDNA.

In another aspect, the invention is directed to a pharmaceuticalformulation. In one embodiment, the pharmaceutical formulation comprisesan rAAV2-hAADC vector and one or more pharmaceutically acceptableexcipients. In one embodiment, the pharmaceutical formulation comprisesa rAAV2-hAADC vector, and 1×PBS. In another embodiment, thepharmaceutical formulation comprises a rAAV2-hAADC vector, 1×PBS, andabout 200 mM NaCl.

Optionally, the pharmaceutical formulation comprises a rAAV2-hAADCvector concentration of at least about 1×10¹¹ vg/mL, at least about5×10¹¹ vg/mL, at least about 1×10¹² vg/mL, or at least about 5×10¹²vg/mL. Preferably, the pharmaceutical formulation comprises arAAV2-hAADC vector concentration of concentration of about 5.7×10¹¹vg/mL.

In another aspect, the invention is directed to compositions and methodsfor increasing AADC gene therapy transduction. In one embodiment, theinvention provides a pharmaceutical formulation comprising anrAAV2-hAADC vector and empty capsids. Optionally, the empty capsids arepresent in a percentage of at least about 0.1% cp/cp, at least about 10%cp/cp, at least about 50% cp/cp, at least about 75% cp/cp, or at leastabout 90% cp/cp.

Optionally, the empty capsids are present in a percentage ranging fromabout 0.1% to about 90% cp/cp, from about 1% to about 90% cp/cp, fromabout 10% to about 80% cp/cp, from about 20% to about 70% cp/cp, fromabout 40% to about 60% cp/cp, from about 10% to about 50% cp/cp, fromabout 10% to about 25% cp/cp, or from about 25% to about 75% cp/cp.Preferably the empty capsids are present in at about 10% vg/vg, about20% vg/vg about 30% cp/cp, about 40% cp/cp, about 50% cp/cp, about 60%cp/cp, about 70% cp/cp, about 80% cp/cp, or about 90% cp/cp. In oneparticular embodiment, the percentage of empty capsids is at least about50% cp/cp. In another particular embodiment, the percentage of emptycapsids is at least about 88% cp/cp. In another particular embodiment,the percentage of empty capsids is about 88% cp/cp. In anotherparticular embodiment, the pharmaceutical formulation comprises about1.76×10¹² cp empty capsids and about 2.4×10¹¹ vg rAAV-hAADC vector.

Optionally, the empty capsids are present in a ratio of empty capsids torAAV2-hAADC vectors of at least about 9 to about 1, at least about 1 toabout 1, or at least about 1 to about 9. Optionally, the pharmaceuticalformulation comprises empty capsids that are present in an excess overrAAV2 hAADC vectors. In one embodiment, the pharmaceutical formulationcomprises empty capsids are present in at least about a 10× excess overrAAV2-hAADC vectors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Biological Substance (rAAV2-hAADC Vector) Diagram.

FIG. 2: Photographs of Surgical Equipment: Insertion tube, Stylet.

FIG. 3: Photographs of Surgical Equipment: Swaged catheter.

FIG. 4: Schematic Drawing of a Stereotactic Arrangement for BilateralInjection into the Putamen.

FIG. 5: Graphical representation of PDMS-2 Total Raw Scores by Subjectand Chronological Age (n=18).

FIG. 6: Graphical representation of LS means and SE for PDMS total scoreover time through 12 months after vector administration (n=18).

FIG. 7: Graphical representation of AIMS Total Scores by Subject andChronological Age (n=18).

FIG. 8: Graphical representation of LS means and SE for AIMS total scoreover time through 12 months after vector administration (n=18).

FIG. 9: Images of ¹⁸F-DOPA PET before and after gene therapy treatment.

FIG. 10: Graphical representation of LS mean PET specific uptake overtime through 12 months after gene therapy treatment.

FIG. 11: Schematic Overview of a rAAV-hAADC Vector ManufacturingProcess.

FIG. 12: Schematic Plasmid Map of pAAV-CMV-hAADC-KanR DNA.

FIG. 13: Nucleotide sequence of a pAAV-CMV-hAADC-KanR plasmid (SEQ IDNO. 1).

FIG. 14: Nucleotide sequence of a rAAV-hAADC-vector gene insert (SEQ IDNO. 2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides several advantages over prior AADC genetherapy. (i) The present invention provides higher doses of rAAV-hAADCvector. (ii) The present invention provides doses of rAAV-hAADC tailoredto a patient's age. (iii) The present invention treats or prevents AADCgene therapy induced dyskinesia. (iv) The present invention increasesAADC gene therapy transduction. (iv) The present invention limitsinterference of a patient's immune system. (v) The present inventionavoids using onerous skull-fixed head frame stereotaxy. (vi) The presentinvention offers greater precision, limited surgical exposure, andgreater safety.

Definitions

The present invention will best be understood with reference to thefollowing terms:

The term “Aromatic L-amino acid decarboxylase” or “AADC” shall mean apolypeptide which decarboxylates dopa to dopamine. (See EC 4.1.1.28;OMIM 107930.) “AADC” includes, but is not limited to, a full-length AADCpolypeptide, active fragment or functional homologue thereof. Literaturereports that AADC is the final enzyme in the biosynthesis of themonoamine neurotransmitters serotonin and dopamine, and dopamine is theprecursor for norepinephrine and epinephrine. See K. et al., “Aromaticamino acid decarboxylase deficiency in twins.” J Inherit Metab Dis.1990; 13(3):301-4.

The term “Aromatic L-amino acid decarboxylase deficiency,” “AADCdeficiency,” or “AADCD” shall mean an inherited disorder of monoamineneurotransmitter syntheses reportedly caused by a homozygous or compoundheterozygous mutation in the AADC gene DDC on chromosome 7p12 (OMIM608643). Literature reports that AADC disorder is clinicallycharacterized by vegetative symptoms, oculogyric crises, dystonia, andsevere neurologic dysfunction, usually beginning in infancy orchildhood. See Brun L, et al., “Clinical and biochemical features ofaromatic L-amino acid decarboxylase deficiency,” Neurology, 2010 Jul. 6;75(1):64-71. Literature also reports that AADC deficiency is anautosomal recessive inborn error in neurotransmitter metabolism thatleads to combined serotonin and catecholamine deficiency. See N. G.Abeling et al. “Pathobiochemical implications of hyperdopaminuria inpatients with aromatic L-amino acid decarboxylase deficiency,” J InheritMetab Dis. 2000 June; 23(4):325-8. Reference is made to Wassenberg etal. “Consensus guideline for the diagnosis and treatment of aromaticL-amino acid decarboxylase (AADC) deficiency” Orphanet Journal of RareDiseases (2017) 12:12. More than 50 different DDC gene disease-causingvariants. A founder splice variant (IVS6+4A>T) is associated with asevere phenotype of AADC deficiency. Medical histories from 37 subjectswith AADC deficiency were reviewed for motor development, mutation, andbody weight. End points for patients were either their latest follow up,death, or entering into a gene therapy clinical trial. The mean age ofthese subjects, at the end points, was 4.78 years (1.31-11.33). Of the37 patients, 36 did not develop a full head control, nor sitting orstanding, during any time point from birth to the end points, and noneof them developed a speech. Their body weights were normal in the firstfew months of life, but severe growth retardation occurred during 1-4years of age. Founder splice variant c.714+4A>T (IVS6+4A>T) represented76% of subjects' DDC mutations.

The term “AAV” shall mean an adeno-associated virus.

The term “AAV2” shall mean an adeno-associated virus serotype 2.

The term “vector” shall mean a vehicle used to deliver genetic materialinto a target cell. A vector can be any genetic element, that is capableof replication when associated with the proper control elements andwhich can transfer gene sequences between cells. Examples of suchgenetic elements, include but are not limited to, a plasmid, phage,transposon, cosmid, chromosome, virus, or virion. The term includes, butis not limited to, cloning and expression vehicles, as well as viralvectors. A vector can optionally be a gene therapy delivery vehicle, orcarrier encapsulating a therapeutic gene for delivery to cells.

The term “recombinant virus” shall mean a virus that has beengenetically altered. Examples of alterations include but are not limitedto, addition or insertion of a heterologous nucleic acid construct.

The term “AAV virus” shall mean a complete virus particle, for example awild-type (wt) AAV virus particle. An AAV virus has an AAV capsidprotein coat encasing a linear, single-stranded AAV nucleic acid genome.An AAV virus is replication-incompetent (i.e., replication-defective orhelper-dependent virus). An intact virus particle may also be referredto as a “virion.” An AAV virus is optionally derived from anyadeno-associated virus serotype, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, or AAVX7. AAV viruses can have one or moreof the AAV wild-type genes deleted in whole or part, preferably the repand/or cap genes, but retain functional flanking ITR sequences. Examplesof AAV viruses include, but are not limited to, AAV viruses that areavailable from the American Type Culture Collection (“ATCC”) underAccession Numbers 53222, 53223, 53224, 53225 and 53226. For adescription of AAV viruses and their uses see, e.g., Haj-Ahmad andGraham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol.67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729;Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al.(1993) Human Gene Therapy 4:461-476.

The term “recombinant AAV,” a “rAAV,” a “recombinant AAV vector,” or a“rAAV vector” shall mean an infectious but replication-defective viruscomposed of an AAV protein shell (i.e., a capsid) encapsulating a geneinsert different from the wild-type AAV DNA.

The term “inverted terminal repeat” or “ITR” shall mean a symmetricalnucleic acid sequence at either end of the genetic material of a virus.Without being bound by theory, literature reports show that ITRs aid theefficient multiplication of the AAV genome. Literature also reports ITRsability to form a hairpin, which contributes to self-priming that allowsprimase-independent synthesis of a second nucleic acid strand. ITRs werealso shown to aid both integration of AAV DNA into a host cell genome.ITRs need not be the wild-type nucleotide sequences, and may be altered,for example, by the insertion, deletion or substitution of nucleotides,so long as the sequences provide for functional rescue, replication andpackaging. Optional nucleotide sequences of AAV ITR regions are known.See, e.g., Kotin, R. M. (1994) “Human Gene Therapy” 5:793-801; Berns, K.I. “Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.)

The term “gene insert” shall mean a nucleic acid molecule encapsulatedby capsid, that codes for a sequence of a polypeptide.

The term “gene therapy” shall mean a treatment of a subject comprisingthe introduction, to a subject, of a normal copy of one or moredefective or missing genes. The term “AADC gene therapy” or “DDC genetherapy” shall mean a treatment of a subject comprising the introductionof a normal copy of a DDC gene to a subject that has a defective DDCgene or is missing a DDC gene.

The term “pediatric AADC gene therapy” shall mean a AADC gene therapytreatment of a minor child subject.

The term “subject,” “individual,” or “patient” shall be usedinterchangeably and shall mean a mammal, preferably a human in need oftherapy. A subject is preferably a minor child aged about 10 years orless. More preferably the subject is aged about 3 years or less.

The term “capsid” shall mean a protein coat or shell of a virus. Acapsid optionally comprises one or more oligomeric structural subunitscomprising proteins, optionally referred to as protomers. A capsid mayoptionally be surrounded by a lipid bilayer and glycoprotein envelope.In one embodiment, a capsid is an adeno-associated virus (AAV) capsid.In one particular embodiment, the capsid is a recombinantadeno-associated virus (rAAV) serotype-2 capsid.

The term “empty capsid” shall mean a virus protein coat that does notcontain a vector genome. An empty capsid can be a virus-like particle inthat it reacts with one or more antibodies that react with intact (e.g.,vector genome carrying) virus (e.g. adeno-associated virus, AAV). In anon-limiting example, an empty AAV2 capsid retains the ability to reactwith one or more antibodies that bind to an AAV, such as an AAV2 oranother AAV serotype. For example, an empty AAV2 capsid retains theability to react with one or more antibodies that bind to AAV8.

Empty capsids may sometimes be naturally found in AAV vectorpreparations. Such preparations can be used in accordance with theinvention. Optionally, such preparations may be manipulated to increaseor decrease the number of empty capsids. For example, the amount ofempty capsid can be adjusted to an amount that would be expected toreduce the inhibitory effect of antibodies. Empty capsids can also beproduced independently of vector preparations, and optionally (i) addedto vector preparations, or (ii) administered separately to a subject.See F. Mingozzi et al., U.S. Patent Application Publication No.2014/0336245 “Virus vectors for highly efficient transgene delivery.”

The term “modified capsid” shall mean a content-modified capsid, or acapsid modified so that the capsid is unable to enter a cell.

The term “content-modified capsid” shall mean a capsid carrying a geneinsert that is modified. Examples of gene inserts that are modified,include but are not limited to, non-coding nucleic acids.

The term “mutant empty capsid” shall mean an empty capsid comprising amutation that disrupts virus receptor binding. In one embodiment, amutant empty capsid is a non-infective mutant capsid. In anotherembodiment, an empty capsid can absorb an antibody but cannot enter atarget cell. In another embodiment, an empty capsid can absorb aneutralizing antibody. See C. J. Aalbers, et al., “Empty Capsids andMacrophage Inhibition/Depletion Increase rAAV Transgene Expression inJoints of Both Healthy and Arthritic Mice,” Human Gene Therapy, 2017February; 28(2):168-1781; and Ayuso E, et al. “High AAV vector purityresults in serotype- and tissue independent enhancement of transductionefficiency.” Gene Ther 2010; 17:503-510.

The term “DDC gene insert” shall mean a gene insert comprising a nucleicacid sequence encoding AADC. In one embodiment, gene insert comprises anucleic acid sequence encoding human AADC (hAADC). In anotherembodiment, the nucleic acid sequence encoding hAADC is an unmodifiedDDC cDNA.

The term “enhancer” shall mean a regulatory sequence, outside thepromoter, that binds transcription factors. An enhancer is optionally aglobin protein, a portion of a globin protein, a human growth hormone, aportion of human growth hormone or a first intron of human growthhormone. Examples of goblin proteins, include but are not limited to,β-globin, human β-globin, human β-globin intron, human β-globin intron2, partial human β-globin intron 2, human 3-globin exon 3, and partialhuman β-globin exon 3. In a preferred embodiment, the enhancer is humanβ-globin partial intron 2/partial exon 3.

The term “plasmid construct” shall mean a circular nucleic acid moleculethat replicates independently of a host cell's genetic material. In oneembodiment, the plasmid construct comprises: a nucleic acid sequenceencoding hAADC, a CMV IEP promoter, an SV40 poly A tail, a humanβ-globin intron 2/exon 3, two ITRs flanking the gene elements at twoends of the gene insert and an antibiotic resistance gene operablylinked to an antibiotic resistance gene

The term “antibiotic resistance gene” shall mean a gene conferringresistance to an antibiotic. Examples of antibiotic resistance genes,include but are not limited, to a resistant gene of ampicillin,kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin,erythromycin, tetracycline, chloramphenicol, and polymyxin B. In oneembodiment, the of antibiotic resistance gene is, a resistant gene ofampicillin. In another embodiment, the of antibiotic resistance gene is,a resistant gene of kanamycin.

The term “vector genome” or “vg” shall be broadly understood toencompass gene inserts. For convenience, “vector genomes” shall include,but shall not be limited to, gene inserts encapsulated within capsidssuch as AAV viruses and rAAV vectors and gene inserts, that are notencapsulated by capsids. For convenience gene inserts, that are notencapsulated by capsids include isolated gene inserts. See U.S. Pat. No.9,598,703 “Capsid-free AAV vectors, compositions, and methods for vectorproduction and gene delivery”

For quantitative purposes, “vg” is calculated as a count of geneinserts. In one example, of a single “vector genome” or single “vg” is asingle gene insert or a single capsid carrying a gene insert. In anotherexample, one rAAV-hAADC vector particle shall mean “1 vg,” while about2.4×10¹¹ vg shall mean about 2.4×10¹¹ rAAV-hAADC vectors.

The term “capsid particle” or “cp” shall be broadly understood toencompass any capsid. For convenience, the capsids may be full (e.g.,encapsulating a gene insert) or empty. Capsid particles include, but notlimited to, capsids carrying vector genomes (e.g., AAV viruses, and rAAVvectors), empty capsids, modified capsids, content-modified capsids, andmutant empty capsids.

For quantitative purposes, “cp” is calculated as a count of the totalnumber of combined capsids carrying vector genomes (e.g., AAV viruses,and rAAV vectors), empty capsids, modified capsids, content-modifiedcapsids, and mutant empty capsids. In one example, “1 cp” shall mean oneempty capsid, while about 1.76×10¹² cp shall mean about 1.76×10¹² emptycapsids. In another example a pharmaceutical formulation comprising 88%cp/cp empty capsids comprises 88 empty capsid particles per 100 totalcapsid particles (full and empty). In another example, a pharmaceuticalformulation can comprise a total of about 2.0×10¹² cp capsid particles,wherein the pharmaceutical formulation comprises about 2.4×10¹¹ vgrAAV-hAADC vectors and about 1.76×10¹² cp empty capsids.

The term “transduction” shall mean the transport of a gene to a cell byusing a virus particle.

The term “effective amount” or “therapeutically effective amount” is anamount sufficient to affect a therapeutically beneficial ortherapeutically desired result. A therapeutically effective amount canbe administered in one or more administrations, applications or dosages.

The term “decoy,” “decoy particle” or “viral decoy” shall mean aparticle or other composition that mimics a virus. The decoy ispreferably devoid of virulent activity. Without being bound by theory, adecoy can mimic a native virus in size, shape, structure, or compositionthereby causing, and thereby can be consumed by macrophages (e.g.,through phagocytosis), leaving functional vectors free to transducecells. Examples of decoy particles include, but are not limited to,empty capsids, modified capsids, and mutant empty capsids.

The term “dyskinesia” shall mean abnormality of voluntary movement,diminished voluntary movement, impairment of voluntary movement, orinvoluntary movement.

The term “target point” or “target site” shall mean a location in thebrain of a subject where the pharmaceutical formulation is administered.Preferably, the target point is the deepest location of injection.

The term “stereotaxy,” “stereotaxis,” or “stereotactic procedure” shallmean a method in neurosurgery or neurological research for locatingpoints within the brain using an external, three-dimensional frame ofreference usually based on the Cartesian coordinate system.

The term “frameless stereotaxy,” “frameless stereotaxis,” or “framelessstereotactic procedure” shall mean stereotaxy performed without a headframe. Particularly, “frameless stereotaxy” uses a surgical supportbracket instead of a skull-mounted head frame. One example of a surgicalsupport bracket is a Nexframe®—Frameless Surgical Support Bracket(Medtronic, Minneapolis, Minn.).

The term “fiducial” or “fiducial marker” shall mean an object used as apoint of reference or a measure. A fiducial is optionally placed into oron a subject where the fiducial appears in the field of view of animaging system and the fiducial appears in the image produced. Afiducial marker may also be used as a bone anchor for a framelessstereotaxy skull platform.

The term “homolog” or “homologous sequence” shall be understood to meana sequence having a percentage identity with the bases of a nucleotidesequence, or the amino acids of a polypeptide sequence, of at leastabout 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.6%, or 99.7%. This percentage is purely statistical, and it ispossible to distribute the differences between the two nucleotide oramino acid sequences at random and over the whole of their length.Sequence identity can be determined, for example, by computer programsdesigned to perform single and multiple sequence alignments.

The term “substantial homology”, when referring to a nucleic acid, orfragment thereof, indicates that, when optimally aligned withappropriate nucleotide insertions or deletions with another nucleic acid(or its complementary strand), there is nucleotide sequence identity inabout 90 to 100% of the aligned sequences.

Embodiments of the Present Invention

The present invention is directed to compositions and methods fortreating aromatic L-amino acid decarboxylase (AADC) deficiency.

In one aspect, the invention is directed to a vector comprising anadeno-associated virus (AAV) containing the human gene encoding the AADCenzyme (hAADC). In one particular aspect, the invention is directed tovector comprising AAV serotype 2 (AAV2) and a DDC gene insert. In oneembodiment, the vector is a rAAV2-hAADC vector comprising an rAAV2capsid, and a complementary DNA (cDNA) sequence encoding hAADC.

The goal of this therapeutic approach is to increase the amount of AADCin target areas of the central nervous system (CNS) to a level that issufficient to result in clinical benefit. Without being bound by anyparticular theory, it is believed that this goal is accomplished byincreasing the production of critical neurotransmitters and subsequentlyimproving neurological function.

In one embodiment, the viral vector of rAAV2-hAADC is an unnatural geneobtained by a genetic engineering by restriction enzyme cleavage and byDNA ligation of human β-globin, where the restriction enzyme cleavage isa cleavage of Cla I, EcoR V, Hind III, Not I, Sac II and Xho I.

The synthesis of an rAAV-hAADC vector may use any current methodologyknown to persons skilled in the art as well as standard variations ofsuch methods. One non-limiting examples of an rAAV-hAADC vector isdescribed in Fan et al., “Behavioral Recovery in6-Hydroxydopamine-Lesioned Rats by Cotransduction of Striatum withTyrosine Hydroxylase and Aromatic L-Amino Acid Decarboxylase Genes UsingTwo Separate Adeno-Associated Virus Vectors”, Human Gene Therapy9:2527-2535, 1998. Other non-limiting examples of rAAV-hAADC vectors aredescribed in C. W. Christine, et al., “Safety and tolerability ofputaminal AADC gene therapy for Parkinson disease,” Neurology 73,1662-1669 (2009); and S. Muramatsu, et al., “A phase I study of aromaticL-amino acid decarboxylase gene therapy for Parkinson's disease,” Mol.Ther. 18, 1731-1735 (2010); K. Ozawa et al. U.S. Pat. No. 7,588,757“Methods of treating Parkinson's disease using recombinantadeno-associated virus virions”; and K. Bankiewicz et al., U.S. Pat. No.6,309,634, “Methods of treating Parkinson's disease using recombinantadeno-associated vector (rAAV); Wuh-Liang Hwu, et al., “Gene Therapy forAromatic L-Amino Acid Decarboxylase Deficiency,” Sci. Transl. Med. 4,134 (2012); and Wuh-Liang Hwu, et al., U.S. Pat. App. Pub. No. US2012/0220648.

Optionally, synthesis of the rAAV-hAADC vector uses HEK293/plasmidtransfection technology. “Production of adeno-associated virus (AAV)serotypes by transient transfection of HEK293 cell suspension culturesfor gene delivery” P. S. Chahal et al., Journal of Virological Methods,2014, Vol. 196, Pages 163-173.

In one embodiment, the gene insert comprises a heterologous nucleic acidand one or more inverted terminal repeats (ITRs). AAV can be constructedusing recombinant techniques that are known in the art and include oneor more heterologous nucleic acids flanked by ITRs.

ITRs are optionally wild-type nucleotide sequences, or nucleic acidsequences that may be altered. Alterations include but are not limitedto, insertion, deletion, or substitution of nucleotides. The sequencescan provide for proper function, including but not limited to, rescue,replication, and packaging of an AAV genome. See for example, “GeneTransfer Vectors for Clinical Application,” Methods in Enzymology;Academic Press, Vol. 507, 1st Ed. (2012).

An example of an rAAV-hAADC vector gene insert is shown in FIG. 1.

//-ITR-CMV-HBG2/3-hAADC-Poly A-ITR-//

-   -   ITR=AAV2 inverted terminal repeat,    -   CMV=cytomegalovirus promoter,    -   HBG2/3=Human Beta Globin Partial intron 2/partial exon 3,    -   hAADC=human aromatic L-amino acid decarboxylase 1.85 kb cDNA.    -   Poly A=SV40 poly-adenylation sequence.

Pharmaceutical Formulations

In another aspect, the invention is directed to a pharmaceuticalformulation. A thorough discussion of pharmaceutically acceptableexcipients is available in “Remington: The Science and Practice ofPharmacy” (22^(11d) Edition, Pharmaceutical Press, New York, N.Y. 2012)

In one embodiment, the pharmaceutical formulation comprises anrAAV2-hAADC vector and one or more pharmaceutically acceptableexcipients.

Optionally, the rAAV2-hAADC vector is formulated in phosphate bufferedsaline (PBS), 1×PBS, 2×PBS, 10×PBS, Dulbecco's PBS (DPBS), or DPBS whichdoes not comprise Magnesium or Calcium.

In one embodiment, the pharmaceutical formulation comprises arAAV2-hAADC vector, and 1×PBS. In another embodiment, the pharmaceuticalformulation comprises a rAAV2-hAADC vector, 1×PBS, and about 200 mMNaCl.

Examples of pharmaceutical formulations include, but are not limited tothe pharmaceutical formulations shown in Table 1.

TABLE 1 Example rAAV2-AADC Formulations Component Formulation 1Formulation 2 Formulation 3 Formulation 4 rAAV2-hAADC 5.67 × 10¹¹ vg/mL1.2 × 10¹² vg/mL 7.41 × 10¹¹ vg/mL 6 × 10¹¹-9 × 10¹¹ vg/mL ExcipientsPBS Dulbecco's PBS Dulbecco's PBS PBS (no Mg, no Ca) + (no Mg, no Ca) +200 mM NaCl 200 mM NaCl pH 7.39 6.99 6.9-7.5

Optionally, the pharmaceutical formulation comprises a rAAV2-hAADCvector concentration of at least about 1×10¹¹ vg/mL, at least about5×10¹¹ vg/mL, at least about 1×10¹² vg/mL, or at least about 5×10¹²vg/mL. Preferably, the pharmaceutical formulation comprises arAAV2-hAADC vector concentration of concentration of about 5.7×10¹¹vg/mL or about 7.5×10¹¹ vg/mL.

Another aspect of the present invention is directed to pharmaceuticalformulations and methods of increasing AADC gene therapy transduction.Vector degradation or vector neutralization, before the vector cantransduce target cells, can decrease vector expression. Decreasedexpression may be caused by vector neutralization by macrophages (e.g.,through phagocytosis or opsonization by soluble factors). Pharmaceuticalformulations optionally comprise agents that influence macrophageactivity or macrophage number, which improves efficacy of recombinantAAV vector transgene expression.

In one particular aspect, decoy particles are administered to a subjectas part of the disclosed method. A non-limiting example of a decoyparticle is an empty capsid. In one particular aspect, empty AAV capsidsare administered to a subject as part of the disclosed method.

Much effort in gene-therapy research has been devoted to minimizing thepresence of empty capsids. Some researchers believe, counterintuitively,that the presence of empty capsids is advantageous for genetransduction. See J. F. Wright, “AAV Empty Capsids: For Better orWorse?” Molecular Therapy 2014 January; 22(1):1-2; D. Grimm, et al.“Titration of AAV-2 particles via a novel capsid ELISA: packaging ofgenomes can limit production of recombinant AAV-2” Gene Therapy, 1999,Volume 6, Number 7, Pages 1322-1330. Without being bound by theory,literature reports that empty capsids act as decoys and thereforeincrease the chances that full virus particles will be able to reachtarget cells. (see C. J. Aalbers, et al., “Empty Capsids and MacrophageInhibition/Depletion Increase rAAV Transgene Expression in Joints ofBoth Healthy and Arthritic Mice,” Human Gene Therapy, 2017 February;28(2):168-178; F. Mingozzi et al., “Overcoming Preexisting HumoralImmunity to AAV Using Capsid Decoys,” Sci. Transl. Med. 2013 July 17;5(194)). Without being bound by theory, literature reports discloseusing viral decoys that are incapable of infectious behavior while atthe same time being fully capable of effecting an immune response andotherwise being antigenically bioreactive. See N. Kossovsky et al., U.S.Pat. No. 5,334,394 “A Human immunodeficiency virus decoy,” and F.Mingozzi et al., U.S. Patent Application Publication No. 2014/0336245“Virus vectors for highly efficient transgene delivery.”

In one embodiment, a pharmaceutical formulation comprises: (a)rAAV2-hAADC vectors, and (b) empty capsids. Optional embodiments includebut are not limited to, pharmaceutical formulations, wherein thepercentage of empty capsids is at least about 0.1% cp/cp, at least about10% cp/cp, at least about 50% cp/cp, at least about 75% cp/cp, or atleast about 90% cp/cp. Another optional embodiment includespharmaceutical formulations, wherein the percentage of empty capsidsranges from about 10% to about 90%. In one particular embodiment, thepercentage of empty capsids is at least about 88% cp/cp. In anotherparticular embodiment, the pharmaceutical formulation comprises about1.76×10¹² cp empty capsids, and about 2.4×10¹¹ vg rAAV-hAADC vector.

Other optional embodiments include, but are not limited to,pharmaceutical formulations, wherein the ratio of empty capsids to rAAV2hAADC vectors is at least about 9:1, is at least about 1:1, is at leastabout 1:9. Other optional embodiments include, but are not limited to,pharmaceutical formulations, wherein the ratio of empty capsids to rAAV2hAADC vectors is any ratio from about 1:1, to about 1:100,000. Otheroptional embodiments include, but are not limited to, pharmaceuticalformulations, wherein the ratio of empty capsids to rAAV2 hAADC vectorsis about 1:10 to 1:100, 1:100 to 1:1000, 1:1000 to 1:10,000, 1:10,000 to1:100,000, or 1:100,000 to 1:>100,000.

Another particular embodiment includes a pharmaceutical formulation,wherein the empty capsids are present in an excess over rAAV2 hAADCvectors. Another particular embodiment, it is useful for empty capsidsto be present in any number from about a 10× excess over rAAV2 hAADCvectors to about 1,000,000× excess over rAAV2-hAADC vectors. Otherparticular embodiments include a pharmaceutical formulation, wherein theempty capsids are present in at least about a 10× excess, 100× excess,or 1,000× excess over rAAV2-hAADC vectors.

In another example, a pharmaceutical formulation can comprise a total ofabout 2.0×10¹² cp capsid particles, wherein the pharmaceuticalformulation comprises about 2.4×10¹¹ vg rAAV-hAADC vector and about1.76×10¹² cp empty capsids.

A non-limiting example of a method for increasing AADC gene therapytransduction comprises the steps of: (a) providing a pharmaceuticalformulation comprising:

(i) an rAAV2-hAADC vector, and (ii) an empty capsid; and (b) deliveringthe pharmaceutical formulation to the brain of the subject.

In another particular aspect, an rAAV2-hAADC vector, is alsoadministered along with an immunosuppressive agent to a subject as partof the disclosed method. A non-limiting example of a method forincreasing AADC gene therapy transduction comprises the steps of: (a)providing a pharmaceutical formulation comprising: (i) an rAAV2-hAADCvector, and (ii) an immunosuppressive agent; and (b) delivering thepharmaceutical formulation to the brain of the subject. Anothernon-limiting example of a method for increasing AADC gene therapytransduction comprises the steps of: (a) providing a pharmaceuticalformulation comprising: (i) an rAAV2-hAADC vector, (ii) an empty capsid,and (iii) an immunosuppressive agent; and (b) delivering thepharmaceutical formulation to the brain of the subject.

The term “immunosuppressive agent” shall mean a composition thatinhibits or prevents activity of a subject's immune system. Withoutbeing bound by theory, literature reports believe that immunosuppressiveagents act by inhibiting the activation and proliferation of immunecells, including macrophages. See Gummert et al., “NewerImmunosuppressive Drugs; A Review,” J. Am. Soc. Nephrol. 10:1366-1380,1999; B. Huang et al. “Advances in Immunotherapy for GlioblastomaMultiforme.” J. Immunol. Res., 2017:3597613.) Examples ofimmunosuppressive agents, include but are not limited to,corticosteroids, glucocorticoids, mineralocorticoids, cytostatics,antibodies, and drugs acting on immunophilins. Examples ofcorticosteroids, include but are not limited to, triamcinolone,triamcinolone acetonide, hydrocortisone, methylprednisolone,prednisolone, prednisone, amcinonide, budesonide, desonide, fluocinoloneacetonide, fluocinonide, halcinonide, and triamcinolone acetonide,beclometasone, betamethasone, dexamethasone, fluocortolone,halometasone, and mometasone. Alclometasone dipropionate, betamethasonedipropionate, betamethasone valerate, clobetasol propionate, clobetasonebutyrate, fluprednidene acetate, and mometasone furoate. ciclesonide,cortisone acetate, hydrocortisone aceponate, hydrocortisone acetate,hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisonevalerate, prednicarbate, and tixocortol pivalate.

Optionally, the immunosuppressive agent is a macrophage depletion agent.In one embodiment, the macrophage depletion agent is a clodronateliposome. Without being bound by theory, literature reports believe thatclodronate liposomes deplete macrophages by inducing apoptosis inphagocytes following phagocyte recognition and uptake. (See, A. Reszkaet al., “Mechanism of action of bisphosphonates,” Current OsteoporosisReports, 1 (2003) 45-52, and R. G. G. Russell, “Bisphosphonates: Thefirst 40 years,” Bone, 49 (2011) 2-19.)

One non-limiting example of a clodronate liposomes includes Clodrosome®liposomal clodronate; (Encapsula NanoSciences LLC Brentwood, Tenn.)Clodronate Disodium Salt, 5 mg/mL, 17 mM; L-α-Phosphatidylcholine, 18.8mg/mL, 24 mM; Cholesterol, 4.2 mg/mL, 11 mM; Suspended in PhosphateBuffered Saline at pH 7.4, multilamellar liposomes (size range of 0.3-3μm). A Clodrosome® Macrophage Depletion Kit (Encapsula NanoSciences LLCBrentwood, Tenn.) comprises a vial of clodronate encapsulated liposomesand a vial of plain liposome for control.

Optionally the clodronate liposomes comprises a mannosylated liposome. Anon-limiting example of a mannosylated liposome comprisesmonoMan3-glutaryl-phosphatidylethanolamine, a phospholipid derivativedisplaying a branched mannose structure on the surface of the liposome.One non-limiting example of a clodronate liposomes kit includes(m-Clodrosome® Mannosylated Clodrosome Macrophage Depletion Kits;Encapsula NanoSciences LLC Brentwood, Tenn.) comprising (a) a vial ofClodronate encapsulated mannosylated liposomes(4-Aminophenyl-alpha-D-Mannopyranoside incorporated into the liposomes:size range (0.3-3 μm) Suspended in Phosphate Buffered Saline at pH 7.4:Clodronate Disodium Salt 5 mg/mL 17 mM; L-α-Phosphatidylcholine 18.8mg/mL 24 mM; Cholesterol 4.2 mg/mL 11 mM;4-Aminophenyl-alpha-D-Mannopyranoside 1 mg/mL 3.69 mM) and (b) a vial ofplain mannosylated liposome for control (m-Encapsome®) (EncapsulaNanoSciences LLC Brentwood, Tenn.).

In one embodiment, an immunosuppressive agent is optionally administeredabout 48 hours before vector administration, about 24 hours beforevector administration, about 12 hours before vector administration,about 6 hours before vector administration. In another embodiment, animmunosuppressive agent is administered at about the same time as thevector administration. In another embodiment, an immunosuppressive agentis administered as a component of the pharmaceutical formulationcomprising the vector. In another embodiment, an immunosuppressive agentis administered at a time ranging from about 6 hours to about 48 hoursafter vector administration.

In one embodiment of the present invention, the stereotactic procedureuses a StealthStation® Integrated Navigation System (Medtronic,Minneapolis, Minn.). (See R. Heermann et al., “Navigation with theStealthStation™ in Skull Base Surgery: An OtolaryngologicalPerspective,” Skull Base. 2001; 11(4): 277-285; and MT Stechison et al.,“A digitized biopsy needle for frameless stereotactic biopsies with theStealthStation,” Neurosurgery. 2000 January; 46(1):239-41; discussion241-2.) In another embodiment, the stereotactic procedure uses a Curve™Image Guided Surgery (BrainLAB, Heimstetten, Germany). (See “NavigatingSurgery: Latest Image-Guided Technology Promises to Lead the Way,”Journal of Clinical Engineering: January/March 2012—Volume 37—Issue 1—p12, the contents of which are incorporated herein it its entirety).Without being bound by theory, StealthStation® and Curve™ Image GuidedSurgery use cameras or electromagnetic fields to capture and relay asubject's anatomy and the surgeon's precise movements in relation to thesubject, to computer monitors in the operating room. Computerizedsystems are used before and during surgery to help orient the surgeonwith three-dimensional images of the subject's anatomy.

Frameless stereotaxy typically uses pre-operative magnetic resonanceimaging (MRI) to patients using surface scalp anatomy or fiducial scalpmarkers. Optionally, Frameless stereotaxy uses a high-fieldintra-operative MRI (iMRI). Without being bound by theory, literaturereports that a patient's scalp may shift slightly between pre-operativeimaging and final surgical positioning with pinion placement, possiblyintroducing error. Frameless stereotaxy using (iMRI) may compensate forsuch errors.

Frameless stereotaxy is commonly understood to use a surgical supportbracket instead of a skull-mounted head frame. In one embodiment of thepresent invention, the surgical support bracket is a Nexframe®—FramelessSurgical Support Bracket (Medtronic, Minneapolis, Minn.). The MedtronicNexframe Stereotactic System is a disposable, frameless, stereotacticguidance system used in conjunction with image-guided surgery (IGS)systems for intracranial surgical procedures. (see R. A. Vega et al.“Image-Guided Deep Brain Stimulation” Neurosurgery Clinics of NorthAmerica, 2014; 25(1):159-172; J. M. Henderson et al., “The applicationaccuracy of a skull-mounted trajectory guide system for image-guidedfunctional neurosurgery.” Comput. Aided Surg. 2004; 9(4):155-60; and KLHolloway, et al., “Frameless stereotaxy using bone fiducial markers fordeep brain stimulation,” J. Neurosurg. 2005; 103(3):404-413.) Anothernon-limiting example of a frameless procedure is Novalis® Radiosurgery(BrainLAB, Heimstetten, Germany). (See R. E. Wurm, et al., “NovalisFrameless Image-Guided Noninvasive Radiosurgery frameless image-guidednoninvasive radiosurgery: Initial Experience,” Neurosurgery (2008) 62(suppl_5): A11-A18.) (See also A. D. Mehta et al., “Framelessstereotactic placement of depth electrodes in epilepsy surgery,” JNeurosurg. 2005 June; 102(6):1040-5).

It is commonly understood that skulls of children are not fullydeveloped and have bone areas that are fragile, uneven or not fused. Inaspects where the surgical support bracket cannot be secured to theskull by screws, the surgical support bracket is held in place by a headholder. A head holder, also referred to as a head clamp or a skullclamp, is commonly understood to be a device used to secure a subject'shead position during surgical procedures. Optionally, the head holdercomprises a flexible arm. The head holder is tightened with a forceappropriate for the subject's skull bone thickness and bone quality. Onenon-limiting example of a head clamp is a Mayfield® Infinity Skull Clamp(Integra LifeSciences Corporation, Plainsboro, N.J.). (See U.S. Pat. No.5,254,079; “Head Clamp.”) Reference is made to the “FDA SafetyCommunication: Neurosurgical Head Holders (Skull Clamps) and DeviceSlippage,” dated Feb. 25, 2016. Reference is also made to C. Berry etal., “Use of cranial fixation pins in pediatric neurosurgery,”Neurosurgery, 2008; 62(4): 913-919; and J. C. Poli et al., “Epiduralhematoma by Mayfield head-holder: case report and review of literature,”Journal of Pediatric Sciences, 2013; 5: e195.

In one embodiment of the present invention, the platform of a Nexframe®Frameless Surgical Support Bracket is held in place against a subject'sskull by a flexible arm operating as a head clamp. Optionally theNexframe® platform is operably attached to the flexible arm using anadapter. Tightening the flexible arm presses the platform against theskull of the subject. In one particular embodiment, the Nexframe® isheld in place against a subject's skull by a Mayfield® Infinity SkullClamp. Optionally the Nexframe® platform is operably attached to theMayfield® Infinity Skull Clamp using an adapter. Optionally any suitableadapter known in the art is used.

In one embodiment, the frameless stereotactic procedure comprises thesteps of: (a) installing one or more bone fiducial markers, (b)installing a skull-mounted platform, (c) drilling a burr hole in a sideof the skull, (d) locating a target point guided by MRI and CT, (e)inserting a guide tube and stylet at or near the target point, (f)removing the stylet and inserting a catheter, and (g) infusing thepharmaceutical formulation.

For administering the pharmaceutical formulation to a second target in aputamen, the frameless trajectory-based stereotactic procedure furthercomprises: (h) locating a second target point guided by MRI and CT, (i)inserting a guide tube and stylet at or near the second target point,(j) removing the stylet and inserting a catheter, and (k) infusing thepharmaceutical formulation.

In one embodiment, the guide tube and stylet are inserted from about 1cm to about 5 cm from a target point. In another embodiment, the guidetube and stylet are inserted about 2 cm from a target point.

In one embodiment, the infusion method includes administration of thepharmaceutical formulation wherein the catheter is withdrawn allowingthe pharmaceutical formulation to be distributed along a tract of about1 mm to about 20 mm. In one particular embodiment, the catheter iswithdrawn allowing the pharmaceutical formulation to be distributedalong a tract of about 5 mm to about 10 mm. In another particularembodiment, the catheter is withdrawn allowing the pharmaceuticalformulation to be distributed along a tract of about 6 mm to about 8 mm.

It is commonly known in the art that a fiducial marker or fiducial is anobject placed in the field of view of an imaging system which appears inthe image produced, for use as a point of reference or a measure. It maybe either something placed into or on the imaging subject, or a mark orset of marks in the reticle of an optical instrument. One or morefiducial markers are useful in conducting the stereotactic proceduredescribed herein. It is useful to use any number of markers from about 2to about 20. See E. M. Thompson et al. “Skull-fixated fiducial markersimprove accuracy in staged frameless stereotactic epilepsy surgery inchildren” J Neurosurg Pediatr. 2011, 7(1):116-9; and C. R. Maurer, Jr.et al. “Registration of Head Volume Images Using Implantable FiducialMarkers” IEEE Transactions on Medical Imaging, 1997, vol. 16, no. 4,447. It is useful to install the fiducial markers in a circular orsquare manner. Optionally, at least about 4, at least about 6, at leastabout 8, at least about 10 fiducial markers are installed. Preferably,at least about 4 fiducial markers are installed. More preferably, atleast about 8 fiducial markers are installed. Optionally, 4, 6, 8, or 10fiducial markers are installed. In one embodiment, 8 fiducial markersare installed in a generally circular manner on the lower part of theskull bones. Without being bound by theory, the fiducial markers areinstalled on the lower part of the skull bones because the lower part ofthe skull are thicker or less fragile.

Fiducial markers our optionally threaded and positioned by screwing thefiducials into a subject's skull. In some embodiments, the fiducials areself-tapping threaded screws. Optionally the fiducials are operablyattached to self-tapping threaded screws. Fiducial markers uponinstallation, preferably avoid regions where the skull bones are toothin or fragile. Without being bound by theory, for children, the riskof skull fracture is pronounced when the bone is less than about 3 mmthick.

Other Fiducial markers are installed by adhering the fiducials to asubject's scalp using an adhesive including, but not limited to, tape orglue.

Fiducial markers may be made of any surgically suitable material. Forexample, fiducial markers optionally comprise stainless-steel, titaniumor gold. In one embodiment, the fiducial markers are stainless-steelfiducial markers.

Fiducial markers are optionally about 1 mm to about 10 mm in length andabout 1 mm to about 10 mm in width.

In one embodiment, the fiducial marker is a stainless-steel self-tappingscrew (about 2.0 mm×about 5.0 mm). Without being bound by theory,stainless steel self-tapping screws provide optimal contrast again thebone and minimize migration. In another embodiment, the fiducial markeris a self-adhesive, Modality Fiducial Marker (about 15 mm outer-diameterand about 3.5 mm thick). (Brain Lab AG, product 52160). Reference ismade to “Principles and Practice of Stereotactic Radiosurgery,” L. S.Chin, Ed. Springer, 2008. Reference is also made to E. M. Thompson, etal., “Skull-fixated fiducial markers improve accuracy in stagedframeless stereotactic epilepsy surgery in children,” Journal ofNeurosurgery: Pediatrics, 2011, 7(1) 116-119; and D. Chen et al.,“Automatic fiducial localization in brain images” International Journalof Computer Assisted Radiology and Surgery, 2006; M Wang et al.,“Automatic localization of the center of fiducial markers in 3D CT/MRIimages for image-guided neurosurgery” Pattern Recognition Letters, 2009,30 (4), 414-420; F. R. Kahn “Deep Brain Stimulation SurgicalTechniques,” Handbook of Clinical Neurology 2013, ch. 3, pg. 27; and K.L. Holloway, “Frameless stereotaxy using bone fiducial markers for deepbrain stimulation,” Journal of Neurosurgery, 2005, 103(3) 404-413.

Delivery of Pharmaceutical Formulation

The pharmaceutical formulation can be delivered by manual injection, byan infusion pumps or by an osmotic pump. Non-manual injection includes,but in not limited to, convection enhanced delivery (CED). Both osmoticand infusion pumps are commercially available from a variety ofsuppliers, for example Alzet Corporation, Hamilton Corporation, Alza,Inc., Palo Alto, Calif.). One non-limiting example of a syringe pump isa Pump 11 Elite Series, Harvard Pump, Harvard Apparatus Holliston, Mass.One non-limiting example of a syringe pump is a Legato™ Syringe Pump, KDScientific Inc. Holliston, Mass.

The pharmaceutical formulation is optionally delivered by catheter andinfusion pump. Any catheter and pump combination suitable for braininfusion is optionally used.

Stereotactic maps and positioning devices are available, for examplefrom ASI Instruments, Warren, Mich. Positioning may also be conducted byusing anatomical maps obtained by CT and/or MRI imaging of the subject'sbrain to help guide the injection device to the chosen target.

Another aspect of the present invention is directed to post-operativeclinical management of dyskinesia. The present invention includesformulations and methods for preventing, ameliorating, treating, orreducing Dyskinesia caused by AADC gene-therapy. In one embodiment, theinvention is directed to method of treating post hAADC gene-therapydyskinesia in a subject comprising: administering a dopamine-antagonistto the subject.

Without being bound by theory, Post Gene-Transduction Dyskinesia isbelieved to be a transient, self-limited, phenomenon. However, thedyskinesia can disturb a patient's sleep. Orofacial dyskinesia caninterfere with swallowing of food or saliva, and may be dangerous if theseverity is high.

Without being bound by theory, the mechanism of dyskinesia is believedto include, but is not limited to, (i) Post-synaptic hypersensitivityand (ii) a Motor learning period. Post-synaptic hypersensitivity:Dopamine receptors become very sensitive after prolonged period ofdopamine deficiency. Post-synaptic hypersensitivity may be the reasonthat younger patients often experienced less severe dyskinesia. Motorlearning period: Purposeful motor movements come from learning, andtherefore the movements of young infants all are dyskinesia. The firstmovements in patients after restoration of dopamine are first dyskinesiaand then gradually transformed to controlled movement, if neuralplasticity remains.

A dopamine-antagonist is optionally administered at or during thefollowing times: (i) simultaneously with gene therapy, (ii) beginning atemergence of dyskinesia symptoms, (iii) beginning at about the beginningof week-4 after gene-transduction, (iv) until the end of about 12 weeksafter gene-transduction, or (v) from about the beginning of week-4 untilabout the end of 12 weeks after gene-transduction.

Examples of dopamine-antagonists include, but are not limited to,clozapine, haloperidol, olanzapine paliperidone, quetiapine risperidone,or ziprasidone. In one particular embodiment, the dopamine-antagonist ishaloperidol or risperidone. In another particular embodiment, thedopamine-antagonist is risperidone.

The dopamine-antagonist optionally may be administered at a dose fromabout 1 mg daily to about 1000 mg daily, at a dose from about 1 mg dailyto about 10 mg daily, or at a dose from about 0.5 mg daily to about 1 mgdaily. In one particular embodiment, the dopamine-antagonist isadministered at a dose of about 0.5 mg per day. In another particularembodiment, the risperidone is administered at a dose of about 0.5 mgper day.

Container:

In one embodiment, the pharmaceutical formulation is contained in apharmaceutical-grade borosilicate glass container with a fluoropolymerlined plastic closure. Examples of fluoropolymers include, but are notlimited to, polytetrafluoroethylene (PTFE) (Teflon®).polyethylenetetrafluoroethylene (ETFE). (Fluorotec®), and a copolymer ofethylene and tetrafluoroethylene (Tefzel®). In one embodiment, theclosure is lined with polytetrafluoroethylene (PTFE) (Teflon®).

Dose:

Reference is made to Wassenberg et al. “Consensus guideline for thediagnosis and treatment of aromatic L-amino acid decarboxylase (AADC)deficiency” Orphanet Journal of Rare Diseases (2017) 12:12.

In one embodiment of the instant invention, the dose per subject rangesfrom about 1.8×10¹¹ vg to about 2.4×10¹¹ vg. In another embodiment, thedose is at least about 2.4×10¹¹ vg, at least about 1×10¹² vg, or atleast about 1×10¹⁴ vg. In another embodiment, the dose is about 1.8×10¹¹vg, about 2.4×10¹¹ vg, or about 1.5×10¹² vg.

The dose is a total dose per subject per administration over all targetsites. For one non-limiting example, a total dose per subject of about1.8×10¹¹ vg includes four injections of about 4.5×10¹⁰ vg (i.e., one4.5×10¹⁰ vg injection at each of two target sites in each of a subject'stwo putamen). For another non-limiting example, a total dose volume persubject of about 320 μL includes four injections of about 80 μL (i.e.,one 80 μL injection at each of two target sites in each of a subject'stwo putamen).

Dose Volume:

In one embodiment, the pharmaceutical formulation is delivered at a dosevolume ranging from about 1 μL to about 1000 μL per target site. Inanother embodiment, the pharmaceutical formulation is delivered at adose volume ranging from about 10 μL to about 100 μL per target site. Inanother embodiment, the pharmaceutical formulation is delivered at adose volume ranging from about 50 μL to about 100 μL per target site. Inone particular embodiment, the pharmaceutical formulation is deliveredat a dose volume of 80 μL per target site.

Dose Concentration:

In one embodiment, the pharmaceutical formulation comprises anrAAV2-hAADC vector concentration of at least about 1×10⁹ vg/mL. Inanother embodiment, the rAAV2-hAADC vector concentration is at leastabout 1×10¹⁰ vg/mL, at least about 1×10¹¹ vg/mL, or at least about1×10¹² vg/mL. In another embodiment, the rAAV2-hAADC vectorconcentration is at least about 5×10¹¹ vg/mL. In one particularembodiment, the pharmaceutical formulation comprises an rAAV2-hAADCvector concentration of about 5×10¹¹ vg/mL.

Rate of Administration:

In one embodiment, the pharmaceutical formulation is delivered at a rateranging from of about 0.1 μL/min to about 10 μL/min.

In another embodiment, the pharmaceutical formulation is delivered at arate of about 1 μL/min, about 2 μL/min, about 3 μL/min, about 4 μL/min,about 5 μL/min, about 6 μL/min, about 7 μL/min, about 8 μL/min about 9μL/min, or about 10 μL/min. In one particular embodiment, thepharmaceutical formulation is delivered at a rate of about 3 μL/min.

In one embodiment, the pharmaceutical formulation, comprising arAAV2-hAADC vector concentration of 5.7×10¹¹ vg/mL, is delivered to at adose volume of about 80 μL per target site at a rate of about 3 μL/min.

Outcome Measures:

Safety data including adverse events, physical and neurologicexaminations, vital signs, clinical laboratory tests, EKGs, brainimaging (CT/MRI), anti-AAV2 antibodies, and viral shedding assay results(blood, urine) are summarized.

Efficacy data for the following clinical outcome measures: developmentaltests for cognitive and motor function, MRI and CT assessments, FDOPAPositron Emission Tomography (PET) putamen-specific radioactivity uptakevalues, CSF neurotransmitter metabolite values, neurological evaluation,viral shedding, and monitoring for anti-AAV2 antibody production.

Motor/Developmental Tests:

The primary efficacy assessments of treatment with AADC gene therapyrely on the use of well-established motor and developmental testsadministered at specified times before surgery, during the first yearafter surgery, and at other times as detailed in the study protocols.

Peabody Developmental Motor Scale (PDMS-II):

The Peabody Developmental Motor Scale, Second Edition (PDMS-II), is askill-based measure of gross and fine motor development for infants andchildren from birth through 5 years of age. This tool separates motordevelopment into gross and fine motor skills. Through a combination ofthe composite scores for the gross and fine motor skills, the examinerhas a reliable estimate of the child's motor skills. It consists of 4gross motor and 2 fine motor subtests, as follows: Reflexes (grossmotor); Stationary (gross motor); Locomotion (gross motor); ObjectManipulation (gross motor); Grasping (fine motor); and Visual-MotorIntegration (fine motor).

Scoring the PDMS-II relies on raw scores, percentiles, standard scores,and age equivalents for the subtests, and quotients for the composites.Raw scores are total points accumulated by a child on a subtest.Developmental ages are often used to convey information to parents ofyoung children. Age equivalents for PDMS-II are called “motor ages”which convey to parents that their child is “passing” on items that achild of a certain chronological age would typically pass. Ageequivalents for PDMS-II subtests are generated from Table C.1 in thePDMS-II manual or by PDMS-II software scoring and report systems.

Alberta Infant Motor Scale:

The Alberta Infant Motor Scale (AIMS) is a 58-item observational measureof infant motor performance for use from birth through the age ofindependent walking (˜18 months). It assesses the sequential developmentof motor milestones in terms of progressive development and integrationof antigravity muscle control. The population intended for assessmentwith AIMS is pre- and full-term infants who are developing typically butare “at risk” due to pre- peri-, or post-natal factors, and who displaytypical patterns of movement, although these may be delayed or immature.The assessment tool is appropriate for identifying infants with allforms of motor delays, including those who are exhibiting immature motordevelopment as well as those who have severe motor delays involvingabnormal patterns of movement. The assessment requires minimal handlingand assesses infant movement in 4 positions: prone, supine, sitting, andstanding.

The AIMS total score is calculated by summing the scores for the 58items with a range of scores between 0 and 58. Higher scores indicatemore mature motor development. The infant's score can then be convertedto a percentile and compared with age-equivalent peers from thenormative sample.

Bayley Scales of Infant and Toddler Development®-Third Edition:

Bayley Scales of Infant and Toddler Development, Third Edition(Bayley-III) offers a standardized assessment of cognitive and motordevelopment for children between 1 and 42 months of age. The assessmentmeasures cognitive, communication, physical, social/emotional, andadaptive areas of development to identify children with developmentaldelays. The test consists of 5 scales of development: Cognitive Scale,Language Scale, Motor Scale, Social Emotional Scale, and AdaptiveBehavior Scale. It is possible to present results for developmental agecorresponding to each subscale vs chronological age.

Comprehensive Developmental Inventory for Infants and Toddlers:

The diagnostic test of the Comprehensive Developmental Inventory forInfants and Toddlers (CDIIT) is one of the child developmental testscovering 5 developmental subtests used for children aged 3 to 72 months.

EXAMPLES

Examples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

AAV2 Vector Manufacture

Viral particles, including but not limited to, vectors, virions, genedelivery vehicles, capsids and empty capsids, useful in the practice ofthe present invention, can be constructed using methods well known inthe art of molecular biology. Viral vectors carrying transgenes can beassembled from polynucleotides encoding the transgene, suitableregulatory elements and elements necessary for production of viralproteins that mediate cell transduction.

Manufacturing Process Summary:

A schematic of the vector manufacturing process is shown in FIG. 7. Thefinal product, Recombinant Adeno-associated Virus Serotype 2 humanL-aromatic amino acid decarboxylase (rAAV2-CMV-hAADC) is usefullyproduced in a campaign mode in compliance with cGMP. The vector isproduced in 293 human embryonic kidney cells using a co-transfectionprocedure. After expansion of 293 cells, which originate from a workingcell bank, into 10-layer CellSTACKS, cells are co-transfected using aCaPO₄ precipitation method with the helper and vector plasmids (pDG-KanRand pAAV-CMV-hAADC-KanR). A schematic plasmid Map of pAAV-CMV-hAADC-KanRDNA is shown in FIG. 8. After approximately 72 hours, cells areharvested. The rAAV2-CMV-hAADC Harvest is frozen and stored.

A representative example of a pAAV-CMV-hAADC-KanR plasmid nucleotidesequence (SEQ ID NO. 1) is shown in FIG. 13. Embodiments of the presentinvention include, but are not limited to functional homologues of thepAAV-CMV-hAADC-KanR plasmid nucleotide sequence (SEQ ID NO. 1).

A representative example of a gene insert is a nucleotide sequencenucleotide sequence (SEQ ID NO. 2) is shown in FIG. 14. The sequencerepresents the pAAV2-CMV-hAADC plasmid from the beginning of the leftITR to the end of the right ITR, i.e. 23-3725. Embodiments of thepresent invention include, but are not limited to functional homologuesof the nucleotide sequence (SEQ ID NO. 2).

Cell harvest is thawed, lysed in 20 mM Tris/150 mM NaCl with 0.5% OctylPhenol Oxylate with Benzonase® and then disrupted by microfluidization.Virions are then purified by Heparin Sepharose affinity chromatography.Peak fractions are collected after elution with Phosphate BufferedSaline (PBS)+200 mM NaCl (HE Intermediate). The HE Intermediate peak isincreased to approximately 1M NaCl and processed further by PhenylSepharose chromatography, and the rAAV2 flow through are collected. Eachof the flow through fractions are diluted with WFI and further purifiedby sulfopropyl cation exchange chromatography and the first peakcollected by elution with PBS+200 mM NaCl (Purified Bulk). The PurifiedBulk is stored frozen for up to one year.

In-process testing is performed on Harvests and Purified Bulk. ThePurified Bulk that meets set specifications are filtered andconcentrated using Hollow Fiber Cartridge ultrafiltration (GEHealthcare) Hollow Fiber Cartridge MWCO=300,000) combined into onecontainer, sterile filtered, and then vialed. Final product testing isperformed on randomly selected final product vials.

Analysis of Full and Empty Capsids

The count of full and empty capsids is, in one embodiment, accomplishedusing transmission electron microscopy and chromatography. Onechromatographic method, which uses a linear gradient elution on CIM QAdisk, assesses charge differences between full and empty capsids (seeBIA Separations 2015, M. Lock, et al.: “Analysis of Particle Content ofRecombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-ExchangeChromatography.” Human Gene Therapy Methods: Part B 23:56-64 (2012)).

Other counting techniques include but are not limited to (i) CsCl oriodixanol gradients, and (ii) electron microscope (EM) assay, totalparticle assay (ELISA) combined with genome copy titration (qPCR).Reference is also made to J. M. Sommer, “Quantification ofAdeno-Associated Virus Particles and Empty Capsids by Optical DensityMeasurement” Molecular Therapy 2003; 7(1):122-128, and D. Grimm, et al.“Titration of AAV-2 particles via a novel capsid ELISA: packaging ofgenomes can limit production of recombinant AAV-2” Gene Therapy, 1999,Volume 6, Number 7, Pages 1322-1330. Methods for separating emptycapsids from full capsids, include but are not limited to, thoseprovided in U.S. Pat. No. 8,137,948, “Methods for Producing Preparationsof Recombinant AAV Virions Substantially Free of Empty Capsids.”

Equipment for Delivering Pharmaceutical Composition:

1) Stainless steel insertion tube (trocar): Approximately 14 gauge×19.4cm long; Stainless Steel Tubing suitable for brain insertion (Medtronic)(FIG. 3)

2) Stainless steel solid stylet that fits into the insertion tube duringits positioning: Removed and replaced by swaged catheter; Solid Stylet;Approximately 1.5 mm outer diameter×19.4 cm long; Solid Stainless Steelsuitable for brain insertion.

3) Swaged Catheter Components: Stainless Steel; Polyimide Tubing(delivers vector solution); Medical Adhesive; Teflon and “Heat-Shrink”Tubing; Microliter Syringe (e.g., Hamilton Syringe, Model 1750 TLL (500μL)). (FIG. 4)

5) Syringe pump: Harvard Pump 11 Elite Nanomite Programmable SyringePump.

Device Description

Vector is delivered by a swaged catheter that is inserted in aprepositioned insertion tube. An insertion tube containing a solidstylet is accurately inserted in a surgery guided by previously-doneMill and CT scans. The insertion tube and stylet are provided in theMedtronic Nexframe® Stereotactic System. Prior to infusion, the catheterand syringe are attached and loaded with vector. The stylet is removedfrom the insertion tube and the swaged catheter is introduced in itsplace. The lengths of the insertion tube and catheter allow the catheterto extend 20 mm beyond the sleeve when fully seated. The vector solutionis precisely administered by a syringe pump connected to the Hamiltonsyringe. The tubing between the catheter and syringe is 100 cm long andflexible enough that the placement of the catheter can be almost totallyindependent of the position of the syringe and Harvard syringe pump.

Example 1 Clinical Study of High Dose AADC Gene Therapy

Eighteen children (˜2 to ˜9 years of age) with severe AADC deficiencywere treated with rAAV2-hAADC vector under two clinical studiesauthorized by the Taiwan FDA (TFDA). Bilateral injections of rAAV2-hAADCvector into the putamen via an established stereotactic procedure haveresulted in remarkable improvements in motor control and achievement ofdevelopmental milestones in many of the children.

A pharmaceutical formulation comprising rAAV2-hAADC vector at aconcentration of 5.7×10¹¹ vg/mL is injected directly into the putamenbilaterally via stereotactic procedure using a commercially availableNeXframe (Medtronic) skull-mounted platform guided by theStealthstation® Treon Navigation System (Medtronic), rAAV2-hAADC vectoris administered at a dose volume of 80 μL per site to four sites (twoper putamen) at a total dose of 1.8×10¹¹ vector genomes (vg) and a totalvolume of 320 μL.

Gene therapy was administered using an established stereotactic surgicaltechnique employed in deep brain stimulation (DBS). DBS is anestablished neurosurgical procedure, with most of its applications inParkinson's disease. Surgery was performed under general anesthesia.

As useful in gene transfer surgery, an injection catheter was usedinstead of the electronic lead used in DBS. Usefully the injectioncatheter was inserted after the placement of the insertion tube into thebrain. A NeXframe skull-mounted platform using bone fiducial markers wasdeveloped specifically to provided precise targeting stability duringframeless trajectory-based procedures. The application of the framelesssystem allowed the ability to perform the operation in children.

Before surgery, magnetic resonance imaging (MRI) was performed, 8stainless steel self-tapping fiducials were then screwed into the skullin a generally circular pattern, avoiding regions where the skull bonesare too thin or fragile. A computed tomography (CT) scan was thenperformed. Using fused MRI and CT images, the trajectories for theinfusion were planned. Reference is made to G. H. Barnett et al.,“Frameless stereotaxy with scalp-applied fiducial markers for brainbiopsy procedures: experience in 218 cases” Journal of Neurosurgery,1999, 91(4) 569-576.

The rAAV2-hAADC vector was injected via a two-track insertion route (twoinjections per putamen). Without being bound by theory, employing amultiple infusion point strategy, administering vector bilaterally toboth putamen, promotes even transduction of the gene to each putamen.The stereotactic surgery was guided by the Stealthstation® TreonNavigation System.

Study AADC-1601: Eight children with severe AADC deficiency wereenrolled and treated. Children enrolled in the study had a diagnosis ofAADC deficiency, defined as decreased HVA and 5-HIAA CSF levels,presence of at least one AADC gene mutation, and presence of clinicalsymptoms. Patients were followed monthly for safety assessments andevery 3 months for efficacy assessments through the first year aftersurgery. Patients returned for assessments every 6 months. The medianage of patients at baseline was 54.0 months (range 24 to 99 months). Themedian age at diagnosis was 15 months (range 4.0 to 29.0 months). Fivepatients were >12 months of age at symptom onset. All patients wereAsian-Other, and 5 were female. Seven patients had homozygous-foundermutation (IVS6+4A>T).

Study AADC 010: Ten children with severe AADC deficiency were enrolledand treated. Patients were followed for at least 1 year. Studyenrollment required a diagnosis of AADC deficiency, defined as decreasedHVA and 5-HIAA CSF levels, elevated L-dopa levels, presence of at leastone AADC gene mutation, and presence of clinical symptoms. Patients werefollowed monthly for safety assessments and every 3 months for efficacyassessments through the first year after surgery. The median age ofpatients at baseline was 34.0 months (range 21 to 102 months). Themedian age at diagnosis was 10.0 months (range 6.0 to 12.0 months). Fivepatients were <6 months of age, and 5 patients were <12 months of age atsymptom onset. Nine patients were Asian-Chinese and 1 was White; thegenders were balanced (5 patients each). Six patients hadhomozygous-founder mutation (IVS6+4A>T).

A recombinant AAV type 2 vector, rAAV2-hAADC, was used in this study.Nucleotide sequence of a pAAV-CMV-hAADC-KanR plasmid (SEQ ID NO. 1) wasused in the manufacture of the rAAV2-hAADC vector. The expressioncassette consisted of a cytomegalovirus immediate-early promoterfollowed by the first intron of human growth hormone, human AADC cDNA,and the simian virus 40 polyadenylation signal sequence. Clinical-graderAAV2-hAADC was manufactured, and quality control was tested incompliance with the current Good Manufacturing Practices. All patientsreceived infusions of the vector into two target points of each putamenby stereotactic surgery. The study was conducted in compliance with theTaiwan Guidelines on Gene Therapy.

Stereotactic Infusion:

The stereotactic surgery was guided by the StealthStation TreonNavigation System (Medtronic). Two target points that were sufficientlydistant from each other in the dorsolateral direction, as confirmed byCT and MRI, were determined for each putamen. One burr hole wastrepanned in each side of the cranial bone, and the vector was injectedvia a two-track insertion route. The vector-containing solution wasprepared at a concentration of 5.7×10¹¹ vg/ml, and 80 μL of the solutionwas injected at a rate of 3 μl/min into each target; the patientreceived 1.8×10¹¹ vg of AAV2-hAADC in total. During the surgery, a guidetube was inserted 2 cm from the target point, located at the middle ofthe depth of putamen. The stylet was removed, and a long catheter wasthen inserted to perform the infusion. During the in-fusion, thecatheter was withdrawn slightly so that the vector could be distributedalong a catheter tract of 6 to 8 mm in length.

Developmental Evaluation:

The AIMS, PDMS-II, and CDIIT scales were used to evaluate the motordevelopment of the patients. The AIMS scale is an observational measureof infant motor performance that can be administered from birth throughthe age at which independent walking occurs. It assesses the sequentialdevelopment of motor milestones. The PDMS-II scale is a skill-basedmeasure of gross and fine motor development for infants and childrenfrom 6 months to 6 years of age that consists of four gross motor andtwo fine motor subtests. The CDIIT scale is a validated tool that isdesigned for infants and children in Taiwan; it is composed of fivesubtests (cognition, language, motor, social, and self-help). The scoreswere rated by a specially trained physical therapist, and the wholeprocess was videotaped. The videos were then evaluated by a blindedclinician to decrease any possible bias.

Pet Study:

AADC expression in the putamen was assessed by PET with FDOPA, which isa substrate for AADC, before surgery and 6 months after the genetransfer. None of the patients took oral carbidopa, a peripheral AADCinhibitor, because they had deficient AADC activity. For the PET study,FDOPA (0.27 mCi/kg, 10 MBq/kg) in saline was infused into theantecubital vein, and a 15-min static acquisition in thethree-dimensional mode was obtained 90 min after the tracer injection.The PET and CT imaging data were acquired and co-registered with a GEPET/CT scanner (Discovery ST-16; GE Healthcare) to produce the fusionimages. The amounts of FDOPA radioactivity observed in the putamen andoccipital lobe were calculated as SUVmax with the Xelerix softwareversion 1.1362 (GE Healthcare). The standardized uptake value (SUV) isdefined as tissue radioactivity concentration divided by the injecteddose divided by the patient weight. SUVmax is the maximum value of SUVin the region of interest drawn around the targeted tissue, that is, theputamen and occipital lobe. Given that there is very little AADC enzymein the occipital lobe, we used the occipital lobe as a measure ofnonspecific background radioactivity. The specific putaminal FDOPAuptake was expressed as the ratio of FDOPA radioactivity in the combined(right and left) putamen (minus non-specific FDOPA uptake by theoccipital lobe) to the radioactivity in the occipital lobe, according tothe following formula:

(putamen−occipital lobe)/occipital lobe.

Analyses of CSF neurotransmitter metabolites and serum anti-AAV2 titers:

The levels of HVA, HIAA, and 3-O-methyldopa in the CSF were measured byhigh-performance liquid chromatography (HPLC) at Medical Neurogenetics.We also measured the L-DOPA levels in the CSF by HPLC (26). The serumlevels of antibodies against AAV2 were determined with an enzyme-linkedimmunosorbent assay (ELISA) (27). The ELISA method used in this studywas developed for the rapid screening of neutralizing antibodies (nAbs).Using whole vector particles as antigens without degradation, weassessed antibodies that were more specific for AAV2, including nAbs.The antibody titers assessed by this ELISA method showed a goodcorrelation with the nAb titers obtained in a previous cell transductionassay.

Family Questionnaire:

To document the symptoms that were more difficult to quantify, parentsof the patients were asked to fill out a questionnaire at the end of thestudy to compare the status before and after the gene transfer. Thequestionnaire contained four items: mood, as a percentage of time in agood versus a bad mood in a day; sweating, in which the scores of 1 to 3indicated normal, increased, and excessive sweating, respectively; bodytemperature, in which the scores of 1 to 3 indicated stable,occasionally unstable, and frequently unstable, respectively; and theseverity of the oculogyric crisis, where scores of 1 to 3 indicatedmild, moderate severe, and very severe, respectively.

Efficacy:

Peabody Developmental Motor Scale 2: The integrated analyses of PDMS 2total and subscale raw scores in studies AADC 1601 and AADC 010demonstrate statistically significant improvement in gross and finemotor skills as early as 6 months after gene therapy. The increase inPDMS 2 total score was driven by statistically significant increases in4 (Stationary, Locomotion, Grasping, and Visual-Motor Integration) ofthe 6 subtests. The significant treatment benefit seen on motor skillsgenerally continued to improve over time. Younger children showed agreater treatment benefit than older children after surgery.

PDMS-2 Total Score: After surgery, patients demonstrated generallycontinuous increases in their PDMS 2 total score (FIG. 6). The leastsquares (LS) mean PDMS total score over time through 12 months after theprocedure is shown graphically in (FIG. 7). The increase in LS mean PDMS2 total score was evident as early as 3 months after the procedure. Theincreases from baseline in LS mean PDMS 2 total score improved over timeand achieved statistical significance by 6 months after the procedure(Table 2). Age at the time of the procedure was a significant factor inthe model with younger patients at baseline showing the greatestincreases in PDMS 2 total score (Table 3).

TABLE 1 Repeated Measures Model for PDMS-2 Total Score - LS Means byTime Point (All Patients n = 18) 95% CI Unadjusted Adjusted Time PointLS Mean (SE) for Mean P-value¹⁾ P-value²⁾ Baseline 7.8 (5.66) −3.5, 19.23 months 23.0 (5.83) 11.3, 34.7 0.0260 0.10410 6 months 41.0 (5.53)29.9, 52.1 <0.0001 <0.0001 9 months 60.5 (6.18) 48.1, 72.9 <0.0001<0.0001 12 months  73.7 (6.14) 61.4, 86.0 <0.0001 <0.0001 ¹⁾Two-sidedp-value for testing H0: Mean Score-Baseline Score = 0 at eachpostbaseline time point. ²⁾Adjusted p-value = min(x*(p-value), 1.0),based on the Bonferroni adjustment. CI = confidence interval; LS = leastsquares; PDMS-2 = Peabody Developmental Motor Scale, Second Edition; SE= standard error.

TABLE 3 Repeated Measures Model for PDMS-2 Total Score - Tests of FixedEffects (All Patients n = 18) Numerator Denominator Fixed Effect DF DFF-value P-value¹⁾ Study ID 1 15.22 0.82 0.3793 Visit 4 58.25 29.99<0.0001 Age at gene 1 14.92 16.98 0.0009 therapy (months) ¹⁾P-value fortesting the significance of each fixed effect in the model. DF = degreesof freedom; ID = identity; LS = least squares; PDMS-2 = PeabodyDevelopmental Motor Scale, Second Edition.

Alberta Infant Motor Scale (AIMS): The integrated analyses of AIMS totaland subscale raw scores in studies AADC 1601 and AADC 010 demonstrateclinically and statistically significant improvement motor functionafter surgery. Increases in AIMS scores were evident as early as 3months after the procedure and improved continuously over a period of 12months. Significant treatment benefit on the Prone, Supine, and Sitsubtest scores contributed to the overall significant treatment benefiton the AIMS total score. Younger children show the most pronouncedtreatment benefit on from surgery.

AIMS Total: After surgery, all patients in the integrated groupdemonstrated generally continuous increases in their AIMS total score(FIG. 7). The LS mean AIMS total score over time through 12 months afterthe procedure is shown graphically in FIG. 8. The increase in LS meanAIMS total score was evident as early as 3 months after the procedure.The increases from baseline in LS mean AIMS total score improved overtime and achieved statistical significance by 6 months after theprocedure (Table 4). Age at the time of the procedure was a significantfactor in the model with younger patients at baseline showing thegreatest increases in AIMS total score (Table 5).

TABLE 4 Repeated Measures Model for AIMS Total Score - LS Means by TimePoint (All Patients n = 18) 95% CI Unadjusted Adjusted Time Point LSMean (SE) for Mean P-value¹⁾ P-value²⁾ Baseline 1.5 (1.81) −2.1, 5.2  3months 5.3 (1.72) 1.8, 8.8 0.0629 0.25157 6 months 10.1 (1.68)  6.7,13.4 <0.0001 0.00020 9 months 14.2 (1.82) 10.5, 17.8 <0.0001 <0.0001 12months  16.9 (1.86) 13.2, 20.6 <0.0001 <0.0001 ¹⁾Two-sided p-value fortesting H0: Mean Score-Baseline Score = 0 at each postbaseline timepoint. ²⁾Adjusted p-value = min(x*(p-value), 1.0), based on theBonferroni adjustment. AIMS = Alberta Infant Motor Scale; CI =confidence interval; LS = least squares; SE = standard error.

TABLE 5 Repeated Measures Model for AIMS Total Score - Tests of FixedEffects (All Patients n = 18) Numerator Denominator Fixed Effect DF DFF-value P-value¹⁾ Study ID 1 15.62 0.13 0.7255 Visit 4 58.67 18.76<0.0001 Age at gene 1 14.90 12.54 0.0030 therapy (months) ¹⁾P-value fortesting the significance of each fixed effect in the model. AIMS =Alberta Infant Motor Scale; DF = degrees of freedom; ID = identity.

¹⁸F-DOPA Positron Emission Tomography (PET):

Analysis of the SUV demonstrated reproducible increases in ¹⁸F-DOPAuptake in the putamen following gene therapy treatment. Signals prior togene therapy were uniformly low in untreated patients. Following genetherapy, ¹⁸F-DOPA PET signals increased in Study AADC 1601 at 6 monthsin 5 of 6 right putaminal measurements and 5 of 6 left putaminalmeasurements, and at 60 months in 10 of 10 right plus left putamenmeasurements. ¹⁸F-DOPA PET signals increased in Study AADC 010 at 12months in 8 of 8 patients for whom measurements were available.Representative images are shown in FIG. 9. After administration of theAAV2-hAADC viral vector, most patients in the integrated groupdemonstrated generally continuous increases in PET specific uptake. TheLS mean PET specific uptake over time through 12 months after theprocedure is shown graphically in FIG. 10. A statistically significantincrease in LS mean PET specific uptake was evident as early as 6 monthsand further increased by 12 months after the procedure.

CONCLUSIONS

Bilateral injection of rAAV-hAADC vector into the putamen resulted indemonstrable and enduring improvements in motor control and achievementof developmental milestones in children with severe AADC. After surgery,patients demonstrated continuous increases in their PDMS 2 total scoresindicating improvement in motor function. In an integrated analysis ofboth studies, improvement on the PDMS 2 total score was evident by 3months after surgery and achieved statistical significance by 6 monthsafter treatment. The results of the AIMS analysis were similar, withpatients showing improvement in motor function from baseline thatachieved statistical significance by 3 months after surgery. Resultsdemonstrate that children with severe AADC deficiency who are treatedearly show a greater treatment benefit than older children at the timeof surgery.

Example 2 A Phase I/II Trial of Gene Therapy for an Inherited Disorderof Monoamine Neurotransmitter Deficiency

Intraputaminal injection of rAAV2-hAADC vector, in a compassionate useprogram, resulted in improvements in the motor function of patients withAADC deficiency. A phase VII trial enrolled 10 AADC deficiency patients(1.7 to 8.4 years) with bilateral intraputaminal injection ofrAAV2-hAADC vector. All stereotactic surgeries and vector injectionswere well tolerated. Patients started to move their arms and mouths 2-3weeks after gene transduction, and new motor skills were observed 2-3months later. At 12 months after gene transduction, all but one patienthad improvements in motor scales, showed increase in cerebral spinalfluid neurotransmitter concentrations, and increase in tracer uptake inFDOPA PET. Anti-AAV2 antibody titers rose in all patients. But titersdecreased a few months after gene transduction. There were no signs ofcerebral or systemic immune reaction during the follow up period.Adverse events related to treatment were generally well tolerated,including events associated with the surgery or transient post-genetransduction dyskinesia. One patient died of influenza B encephalopathy10 months after gene transduction, but his 9-month motor scales hadshown improvement. Preliminary evidence showed more substantialimprovements in motor and cognitive function in the youngest patientstreated. In conclusion, rAAV2-hAADC gene therapy is a potentialtreatment for an inherited brain neurotransmitter deficiency, andtreatment at a younger age may be associated with a better outcome.

Example 3

Gene Therapy for Aromatic L-Amino Acid Decarboxylase Deficiency: 5 Yearsafter rAAV2-hAADC Transduction

Injection of rAAV2-hAADC vector bilaterally (i.e., to both putamens ofsubjects) with AADC deficiency resulted in improvements in patients'motor function. Data was obtained from 5 patients who have been followedfor more than 5 years after treatment. These patients did not have headcontrol and had not achieved other major motor milestones prior to genetransduction, but started to gain new motor skills after genetransduction. Motor development and cognitive function exhibitedimprovement over this 5-year period, with the most substantial gainsobserved during the first two years after gene transduction. At 5 yearsafter gene transduction, FDOPA PET still exhibited signals of AADCactivity over the putamens. Patients' anti-AAV2 antibody titers roseafter gene transduction, peaked a few months later, and then decreased.There were no signs of cerebral or systemic immune reaction during thefollow up period. Therefore, treatment with rAAV2-hAADC demonstratesencouraging evidence of long-term safety and therapeutic efficacy forpatients with AADC deficiency.

Example 4 Treatment of Post Gene-Transduction Dyskinesia

Patients who received gene-transduction for AADC deficiency experiencedpost gene-transduction dyskinesia characterized by symptoms includingexaggerated and uncontrolled movements over the mouth, face, andextremities. Patients were evaluated from 1-3 months after genetransduction. Administration of risperidone, a suitable dopamineantagonist resulted in reduced or elimination of dyskinesia whileallowing motor development.

Dose Regimen:

Risperidone (1 mg/mL oral solution) was administered 0.1 mL-0.2 mL twicedaily (BID).

Patients in the Phase I/II trial: AADC001-AADC008:

AADC001: Patient received gene transduction at the age of 6 years.Dyskinesia occurred at 5 weeks after gene transduction. Dyskinesiaincreased thereafter. Risperidone was administered beginning at the 7thweek. Dyskinesia remained high for 3 weeks and then went down.Medication stopped 12 weeks after gene transduction.

AADC002: Patient received gene transduction at the age of 7.5 years.Risperidone treatment started when dyskinesia occurred at 4 weeks aftergene transduction. Dyskinesia became more prominent and the dosage ofthe medication was increased. Dyskinesia then decreased and themedication stopped 12 weeks after gene transduction.

AADC003: Patient received gene transduction at the age of 8.5 years.Risperidone treatment started when dyskinesia occurred at 5 weeks aftergene transduction. Dyskinesia became more prominent and we needed toincrease the dosage of the medication. Dyskinesia then decreased and themedication was stopped 10 weeks after gene transduction.

AADC004: Patient received gene transduction at the age of 2.5 years.Dyskinesia was mild and transient. Patient did not receive dopamineantagonist treatment.

AADC005: Patient received gene transduction at the age of 2.5 years.Dyskinesia was mild and transient. Patient did not receive dopamineantagonist treatment.

AADC006: Patient received gene transduction at the age of 6.5 years.Risperidone treatment started when dyskinesia occurred at 6 weeks aftergene transduction and interfered with the patient's swallowing.Dyskinesia became more prominent and the dosage of the medicationincreased twice. Dyskinesia then decreased and the medication stopped 10weeks after gene transduction.

AADC007: Patient received gene transduction at the age of 2.5 years.Dyskinesia was mild. Risperidone treatment started at the 6th week andstopped 8 weeks after gene transduction.

AADC008: Patient received gene transduction at the age of 3 years.Dyskinesia was moderate. Risperidone treatment started at the 4th week,increasing the dose once, and stopped treatment 8 weeks after genetransduction.

Patients in the compassionate use trial: CU001-CU008 (In this earlystudy, the dopamine antagonist treatment used to treat post-genetransduction dyskinesia was not planned. Only patients CU003 & CU008received dopamine antagonist treatment.

CU001: Patient received gene transduction at the age of 4 years.Patient's post gene-transduction dyskinesia, occurred 2-3 months aftergene transduction, was mild. Orofacial dyskinesia seemed to inducenausea, and the parents had given the patient anti-emetic agents.

CU002: Patient received gene transduction at the age of 4.5 years.Orofacial dyskinesia started one month after gene transduction whichsometimes interfered with his saliva swallowing. For one month, hiscondition was difficult due to choking and infection. At two monthsafter gene transduction, his tongue movement was still exaggerated, butthe interference on saliva swallowing decreased. Orofacial and limbdystonia persisted for a few months, but his general condition wasstable. The parents didn't bring him back to the clinic after theone-year clinical trial period.

CU003: Patient received gene transduction at the age of 4.5 years.Orofacial dyskinesia disturbed her saliva swallowing one month aftergene transduction. The parents needed to aspirate her saliva almostevery several minutes, and feeding was administrated through NG andsleep was poor. Anti-emetic medication was not effective on herdyskinesia. Risperidone was prescribed. The dose was increased over twoweeks. This medication was effective and choking by saliva decreased.However, her movements over the extremities were also suppressed, so weneeded to stop this medication. The suppressive effect lasted for amonth after the last dosing. Three months after gene transduction, hermotor movements returned gradually. Orofacial dyskinesia persisted for 5months.

CU004: Patient received gene transduction at the age of 6 years.Orofacial dyskinesia started one month after gene transduction but wasnot serious. No specific medication was given. Three months after genetransduction, she could eat semi-liquid foods by mouth.

CU005: Patient received gene transduction on at the age of 2 years.Dyskinesia occurred one month after gene transduction, but was mild:some mouth movements and intermittent choreoathetoid movements ofextremities. The dyskinesia was self-limited and no specific managementswere given.

CU006: Patient received gene transduction, at the age of 2.5 years. 3weeks after gene transduction, she had choreoathetoid movements of themouth and fingers. Dyskinesia became most severe 6 weeks after genetransduction, which interfered with her sleep and swallowing. Theseverity of dyskinesia then decreased and no specific medication wasgiven to her.

CU007: Patient received gene transduction at the age of 6.5 years. Hisgeneral condition was very poor, and he had tracheostomy, gastrostomy,and anemia (parents refuse transfusion).

CU008: Patient received gene transduction on at the age of 8 years.Orofacial dyskinesia appeared one month after gene transduction, andRisperidone was prescribed. Dyskinesia increased a little bitthereafter, and then decreased. The medication was used for only twoweeks.

Example 5

Age-Determined Dose Treatment with rAAV2-hAADC Vector

Using frameless stereotaxy, 80 μL of rAAV2-hAADC is injected into fourtarget points in a patient's putamen at a rate of 3 μL/min, where therAAV2-hAADC has a concentration of 5.7×10¹¹ vg/mL. A total of 1.8×10¹¹vg of the viral vector is injected.

The AAV viral vector delivers AADC genes into putamens in striatums oftwo AADC deficiency patients. The first patient is a 4-year-old girl whohad profound hypotonia and lack of any motor development. After thetreatment, activities of the limbs are increased, control of the limbsis improved and recognition is enhanced. The girl can sit well withoutsupport with good head control and touch things with her hands one yearafter the treatment. The second patient is a 3.5-year-old boy. Twomonths after the treatment, activities of the limbs are increased andcontrol of the trunk is also improved. In conclusion, the presentdisclosure uses the AAV viral vectors to transfer AADC genes fortreating AADC deficiency practically and effectively.

Example 6

Treatment with High Dose rAAV2-hAADC Vector

Six children with severe AADC deficiency are enrolled and treated.Children enrolled in the study have a diagnosis of AADC deficiency,defined as decreased HVA and 5-HIAA CSF levels, presence of at least oneAADC gene mutation, and presence of clinical symptoms. Patients arefollowed monthly for safety assessments and every 3 months for efficacyassessments through the first year after surgery. Patients return forassessments every 6 months. The six children have homozygous-foundermutation (IVS6+4A>T).

Using frameless stereotaxy, 80 μL of rAAV2-hAADC are injected into fourtarget points in a patient's putamen (320 μL total) at a rate of 3μL/min, where the rAAV2-hAADC has a concentration of 7.5×10¹¹ vg/mL. Atotal of 2.4×10¹¹ vg of the viral vector is injected.

Integrated analyses of PDMS-2 total and subscale raw scores demonstratestatistically significant improvement in gross and fine motor skills asearly as 6 months after gene therapy. An increase in PDMS-2 total scoreis driven by statistically significant increases in 4 (Stationary,Locomotion, Grasping, and Visual-Motor Integration) of the 6 subtests. Asignificant treatment benefit seen on motor skills generally continuesto improve over time. After surgery, patients demonstrate generallycontinuous increases in their PDMS-2 total score. The increase in LSmean PDMS-2 total score is evident as early as 3 months after theprocedure. The increases from baseline in LS mean PDMS-2 total scoreimprove over time and achieve statistical significance by 6 months afterthe procedure.

One patient is a 2-year-old girl who has profound hypotonia and lack ofany motor development. After the treatment, activities of the limbs areincreased, control of the limbs is improved and recognition is enhanced.The girl can sit well without support with good head control and touchthings with her hands one year after the treatment. A second patient isa 1.5-year-old boy. Two months after the treatment, activities of thelimbs are increased and control of the trunk is also improved. Inconclusion, the present disclosure uses the AAV viral vectors totransfer AADC genes for treating AADC deficiency practically andeffectively.

Example 7

Treatment with rAAV2-hAADC Vector and Empty Capsids

Using frameless stereotaxy, 80 μL of a pharmaceutical formulationcomprising about 2.4×10¹¹ vg rAAV2-hAADC vector and about 1.76×10¹²empty capsids, is injected into four target points in a patient'sputamen at a rate of 3 μL/min. The AAV viral vector delivers AADC genesinto putamens in striatums of two AADC deficiency patients. Integratedanalyses of PDMS-2 total and subscale raw scores demonstratestatistically significant improvement in gross and fine motor skills asearly as 6 months after gene therapy. An increase in PDMS-2 total scoreis driven by statistically significant increases in 4 (Stationary,Locomotion, Grasping, and Visual-Motor Integration) of the 6 subtests. Asignificant treatment benefit seen on motor skills generally continuesto improve over time. After surgery, patients demonstrate generallycontinuous increases in their PDMS-2 total score. The increase in LSmean PDMS-2 total score is evident as early as 3 months after theprocedure. The increases from baseline in LS mean PDMS-2 total scoreimprove over time and achieve statistical significance by 6 months afterthe procedure. The first patient is a 4-year-old girl who had profoundhypotonia and lack of any motor development. After the treatment,activities of the limbs are increased, control of the limbs is improvedand recognition is enhanced. The girl can sit well without support withgood head control and touch things with her hands one year after thetreatment. The second patient is a 5-year-old boy. Two months after thetreatment, activities of the limbs are increased and control of thetrunk is also improved. In conclusion, the present disclosure uses theAAV viral vectors to transfer AADC genes for treating AADC deficiencypractically and effectively.

What is claimed is:
 1. A method of treating AADC deficiency in apediatric subject, comprising the steps of: (a) providing apharmaceutical formulation comprising an rAAV2-hAADC vector, (b)stereotactically delivering the pharmaceutical formulation to at leastone target site in the brain of the subject in a dose of an amount atleast about 1.8×10¹¹ vg; wherein delivering the pharmaceuticalformulation to the brain is by frameless stereotaxy.
 2. The method ofclaim 1, wherein the dose is an amount of at least about 2.4×10¹¹ vg. 3.The method of claim 1, wherein the pharmaceutical formulation comprisesa rAAV2-hAADC vector concentration of about 5.7×10¹¹ vg/mL.
 4. Themethod of claim 1, wherein the pharmaceutical formulation is deliveredat a rate of about 3 μL/min.
 5. The method of claim 1, wherein thepharmaceutical formulation is delivered to at least one target site in abrain at a dose volume of about 80 μL per target site.
 6. The method ofclaim 1, wherein the rAAV2-hAADC vector comprises: a) a WT AAV2 capsid,and b) a recombinant DNA DDC gene insert comprising: (i) a firstinverted terminal repeat (ITR), (ii) a cytomegalovirus (CMV) immediateearly promoter (IEP)IEP, (iii) a human β-globin partial intron2/exon 3,(iv) a nucleic acid sequence encoding hAADC, (v) an SV40 poly A tail,and (vi) a second ITR; wherein the first ITR and second ITR flank theCMV IEP promoter and the Poly A tail.
 7. The method of claim 6, whereinthe nucleic acid sequence encoding hAADC is an unmodified DDC cDNA. 8.The method of claim 1, wherein the pharmaceutical formulation isdelivered to a putamen of the brain.
 9. The method of claim 1, whereinthe pharmaceutical formulation is delivered bilaterally to each putamen.10. The method of claim 9, wherein said bilateral delivery is to pointsabout 1 mm to about 10 mm apart.
 11. A method of treating AADCdeficiency in a pediatric subject, comprising the steps of: (a)providing a pharmaceutical formulation comprising an rAAV2-hAADC vector,(b) stereotactically delivering the pharmaceutical formulation to atleast one target site in the brain of the subject in a dose of an amountat least about 2.4×10¹¹ vg.
 12. A method of treating AADC deficiency ina pediatric subject aged less than about 3 years, comprising the stepsof: (a) providing a pharmaceutical formulation comprising an rAAV2-hAADCvector, (b) stereotactically delivering the pharmaceutical formulationto at least one target site in the brain of the subject in a dose of anamount at least about 2.0×10¹¹ vg.
 13. The method of claim 12, whereinthe dose is about 2.4×10¹¹ vg per subject.
 14. A method of treating AADCdeficiency in a pediatric subject aged about 3 or more years, comprisingthe steps of: (a) providing a pharmaceutical formulation comprising anrAAV2-hAADC vector, (b) stereotactically delivering the pharmaceuticalformulation to at least one target site in the brain of the subject in adose from about 1.8×10¹¹ vg to about 2.4×10¹¹ vg.
 15. The method ofclaim 14, wherein the dose is about 1.8×10¹¹ vg.
 16. The method of claim1, further comprising the step of: (c) administering a therapeuticallyeffective dose of dopamine-antagonist to the subject.
 17. The method ofclaim 16 wherein the dopamine-antagonist is clozapine, haloperidol,olanzapine paliperidone, quetiapine risperidone, or ziprasidone.
 18. Themethod of claim 16 wherein the dopamine-antagonist is administered at adose from about 0.1 mg daily to about 1000 mg daily.
 19. A method oftreating AADC deficiency in a pediatric subject, comprising the stepsof: (a) providing a pharmaceutical formulation comprising an AAV2-hAADCvector, (b) delivering the pharmaceutical formulation to the brain ofthe subject, and (c) administering a therapeutically effectivedopamine-antagonist to the subject.
 20. The method of claim 19, whereinthe dopamine-antagonist is administered from about the beginning ofweek-4 after gene-transduction until at least about the end of 12-weeksafter gene-transduction.
 21. The method of claim 19, wherein thedopamine-antagonist is clozapine, olanzapine paliperidone, quetiapine,risperidone, or ziprasidone.
 22. The method of claim 19, wherein thedopamine-antagonist is administered at a dose from about 0.1 mg daily toabout 1000 mg daily.
 23. A pharmaceutical formulation comprising: (a) anrAAV2 hAADC vector, and (b) 1×PBS.
 24. The pharmaceutical formulation ofclaim 23, wherein the pharmaceutical formulation further comprises: (c)about 200 mM NaCl.
 25. The pharmaceutical formulation of claim 23,wherein the pharmaceutical formulation further comprises rAAV2 hAADCvector at a concentration of about 5.7×10¹¹ vg/mL.
 26. Thepharmaceutical formulation of claim 23, wherein the pharmaceuticalformulation further comprises: (d) empty capsids at a percentage of atleast about 0.1% cp/cp.